Induced activation in dendritic cells

ABSTRACT

The present invention is directed to a composition and method which to treat diseases and to enhance a regulated immune response. More particularly, the present invention is drawn to compositions that are based on dendritic cells modified to express an inducible form of a co-stimulatory polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/165,360, filed Jun. 30, 2008, and entitledINDUCED ACTIVATION IN DENDRITIC CELLS, naming David Spencer, BrentHanks, and Kevin Slawin as inventors, which is a continuationapplication of allowed U.S. patent application Ser. No. 10/781,384,issued as U.S. Pat. No. 7,404,950, filed on Feb. 18, 2004, which is anon-provisional patent application claiming priority to U.S. ProvisionalApplication No. 60/448,046 filed Feb. 18, 2003, which are all referredto and all incorporated by reference herein in their entirety. Thisapplication incorporates by reference the computer readable “SequenceListing” that was filed on Feb. 18, 2004, in U.S. patent applicationSer. No. 10/781,384, filed Feb. 18, 2004.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made in part with government support under Grant No.PC010463 awarded by the Department of Defense. The United StatesGovernment may have certain rights in the invention.

TECHNICAL FIELD

The present invention is drawn to compositions and methods to enhance animmune response. More particularly, the composition is an inducibleco-stimulatory polypeptide and is induced by ligand oligomerization.

BACKGROUND OF THE INVENTION

Dendritic cells (DC) are unique among antigen-presenting cells (APC) byvirtue of their potent capacity to activate immunologically naive Tcells (Steinman, 1991). DC express constitutively, or after maturation,several molecules that mediate physical interaction with and deliveractivation signals to responding T cells. These include class I andclass II MHC molecules CDSO (B7-1) and CD86 (B7-2); CD40; CD11a/CD18(LFA-1); and CD54 (ICAM-1) (Steinman, 1991; Steinman et al., 1995). DCalso secrete, upon stimulation, several T cell-stimulatory cytokines,including IL-1-beta, IL-6, IL-8, macrophage-inflammatory protein-1-alpha(MIP-1-alpha), and MIP-1-delta (Matsue et al., 1992; Kitajima et al.,1995; Ariizumi et al., 1995; Caux et al., 1994; Heufler et al., 1992;Schreiber et al., 1992; Enk et al., 1992; Mohamadzadeh et al., 1996).Both of these properties, adhesion molecule expression and cytokineproduction are shared by other APC (e.g., activated macrophages and Bcells), which are substantially less competent in activating naive Tcells.

T cell activation is an important step in the protective immunityagainst pathogenic microorganisms (e.g., viruses, bacteria, andparasites), foreign proteins, and harmful chemicals in the environment.T cells express receptors on their surfaces (i.e., T cell receptors)that recognize antigens presented on the surface of antigen-presentingcells. During a normal immune response, binding of these antigens to theT cell receptor initiates intracellular changes leading to T cellactivation. DC express several different adhesion (and co-stimulatory)molecules, which mediate their interaction with T cells. Thecombinations of receptors (on DC) and counter-receptors (on T cells)that are known to play this role include: a) class I MHC and CD8, b)class II MHC and CD4, c) CD54 (ICAM-1) and CD11a/CD18 (LFA-1), d) ICAM-3and CD11a/CD18, e) LFA-3 and CD2, f) CD80 (B7-1) and CD28 (and CTLA4),g) CD86 (B7-2) and CD28 (and CTLA4) and h) CD40 and CD40L (Steinman etal., 1995) Importantly, not only does ligation of these moleculespromote physical binding between DC and T cells, it also transducesactivation signals.

The dendritic cell (DC) orchestrates several critical steps in thedevelopment of an adaptive immune response. DCs communicate informationregarding the antigenic state of the peripheral tissues to the locallymph nodes. Upon detection of both pathogen-derived and endogenous“danger signals”, the DC physiologically adapts to its microenvironmentby undergoing a genetic program known as “maturation” in order to directan effective T cell response. The unique machinery of the DC allows it,not only to induce the activation of naive T cells, but also to regulatetheir subsequent phenotype and function. These impressive attributesmake the DC an ideal choice for their exploitation as natural adjuvantsin cancer vaccine development. However, the limited successes of recentclinical trials indicate that current DC therapeutic strategies are inneed of further refinement if DC immunotherapy is to be included in thecancer treatment arsenal alongside the more traditional modalities ofchemo- and radiotherapy. This translation of DC vaccine development intothe clinic will rely significantly upon advancements in ourunderstanding of basic DC biology.

One of the critical deficiencies of DC-based vaccines is their transientnature. The activation state and the longevity of DCs are significantlylimited. Less than 24 hours following exposure to bacteria-derivedlipopolysaccharide (LPS), DCs terminate synthesis of the IL-12 cytokineand become refractory to further stimuli. This implies that thecytotoxic T lymphocyte (CTL) activation potential of DCs is severelycompromised a relatively short time following its activation. Vaccinestudies indicate that the survival of antigen-pulsed DCs within thedraining lymph node is dramatically reduced 48 hours following theirdelivery and undetectable by 72 hours. These findings justify the needfor alternative strategies for DC vaccine design, such as thedevelopment of genetically altered DCs that can circumvent physiologicalregulatory mechanisms and exhibit enhanced immunostimulatory propertiesfor the treatment of cancer and other diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a composition and method thatinduces and/or activates antigen-presenting cells. The activatedantigen-presenting cells can be used to enhance and/or regulate immuneresponses to a target antigen. More particularly, the present inventionis drawn to compositions that are based on dendritic cells modified invivo or ex vivo to express an inducible form of a co-stimulatorypolypeptide molecule. The compositions of the present invention can beused to bolster the immune response of an immunocompromised subject,such as an HIV-infected subject. In certain embodiments, the presentinvention utilizes the power of CID to dimerize the co-stimulatorypolypeptide.

Certain embodiments of the present invention include an expressionconstruct comprising a polynucleotide promoter sequence, apolynucleotide sequence encoding a co-stimulatory polypeptide and apolynucleotide sequence encoding a ligand-binding region, alloperatively linked. It is envisioned that the expression construct iscomprised within a vector forming an expression vector; the vector isselected from the group consisting of a viral vector, a bacterial vectorand a mammalian vector. Co-stimulatory polypeptides include, but are notlimited to Pattern Recognition Receptors, C-reactive protein receptors(i.e., Nod1, Nod2, PtX3-R), TNF receptor (i.e., CD40, RANK/TRANCE-R,OX40, 4-1BB), and HSP receptors (Lox-1 and CD-91). In certainembodiments of the present invention, the expression construct and/orexpression vector can be administered to a subject to ehance an immuneresponse in the subject or bolster the immune response in the subject.

The expression construct may further include a second ligand-bindingregion, in which the ligand-binding region is a small molecule-bindingdomain, for example a FKBP binding domain. Yet further, the expressionvector further comprises a polynucleotide sequence encoding a membranetargeting sequence, for example myristoylation-targeting sequence. Incertain embodiments, the polynucleotide promoter sequence is selectedfrom the group consisting a constitutive promoter (i.e., simian virus 40(SV40) early promoter, a mouse mammary tumor virus promoter, a humanimmunodeficiency virus long terminal repeat promoter, a Moloney viruspromoter, an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, a human actionpromoter, a human myosin promoter, a human hemoglobin promoter,cytomegalovirus (CMV) promoter, an EF1-alpha promoter, and a humanmuscle creatine promoter) an inducible promoter (i.e., metallothioneinpromoter, a glucocorticoid promoter, a progesterone promoter, and atetracycline promoter) and a tissue specific promoter (i.e., dendriticcell (i.e., CD11c), PSA associated promoter or prostate-specificglandular kallikrein).

Other embodiments of the present invention comprise a transduced cell,in which the cell is transduced with the expression vector and/orexpression construct of the present invention. More specifically, thecell is an antigen-presenting cell or an embryonic stem cell. It iscontemplated that the transduced cell can be a pharmaceuticalcomposition.

Other embodiments of the present invention include a fusion cellcomprising a transduced antigen-presenting cell fused to a cell, whereinthe transduced antigen-presenting cell comprises an expression vectorand/or expression construct. More specifically, the cell is a tumorcell, for example a prostate tumor cell. It is contemplated that thefusion cell can be a pharmaceutical composition.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising the expression vector or expression construct anda pharmaceutically acceptable carrier, wherein said expression vectorcomprises a polynucleotide promoter sequence, a first polynucleotidesequence encoding a ligand-binding region, a second polynucleotidesequence encoding a ligand-binding region, a membrane-targetingsequence, and a polynucleotide sequence encoding a co-stimulatorypolypeptide, all operatively linked.

Further embodiments of the present invention comprise a method ofactivating an antigen-presenting cell comprising the step of transducingthe antigen-presenting cell with an expression vector, wherein theexpression vector comprises a polynucleotide promoter sequence, apolynucleotide sequence encoding a ligand-binding region, and apolynucleotide sequence encoding a co-stimulatory polypeptide, alloperatively linked; and activating the transduced antigen-presentingcell with ligand resulting in oligomerization. The co-stimulatorypolypeptide includes, but is not limited to Pattern RecognitionReceptors, C-reactive protein receptors (i.e., Nod1, Nod2, PtX3-R), TNFreceptor (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), and HSP receptors(Lox-1 and CD-91). More specifically, the co-stimulatory polypeptide isa CD40 cytoplasmic domain.

A further embodiment of the present invention comprises a method ofmodulating an immune response in a subject comprising the step ofadministering to the subject an expression vector of the presentinvention. The expression vector is expressed in dendritic cells and thevector comprises a polynucleotide promoter sequence, a polynucleotidesequence encoding a ligand-binding region, and a polynucleotide sequenceencoding a co-stimulatory polypeptide, all operatively linked. Thesubject in whom the expression vector can be administered can be asubject that is immunocompromised.

Another embodiment comprises a method of modulating an immune responsein a subject comprising the steps of: transducing an antigen-presentingcell with an expression vector, wherein the expression vector comprisesa polynucleotide promoter sequence, a polynucleotide sequence encoding aligand-binding region, and a polynucleotide sequence encoding aco-stimulatory polypeptide, all operatively linked; and administering tothe subject transduced antigen-presenting cells, wherein the transducedantigen-presenting cells enhance the immune response in the subject. Thetransduced antigen-presenting cell is activated by administering aligand that results in oligomerization. It is further envisioned thatthe transduced antigen present cells are administered to the subjectsimultaneously or subsequently to administration of an immunogeniccomposition.

Another embodiment of the present invention is a method of inducing aregulated immune response against an antigen in a subject comprising thesteps of: transducing an antigen-presenting cell with an expressionvector, wherein the expression vector comprising a polynucleotidepromoter sequence, a polynucleotide sequence encoding a ligand-bindingregion, and a polynucleotide sequence encoding a co-stimulatorypolypeptide, all operatively linked; loading transducedantigen-presenting cells with the antigen; administering transduced,loaded antigen-presenting cells to the subject thereby effecting acytotoxic T lymphocyte and natural killer cell anti-tumor antigen immuneresponse; and regulating the immune response induction directed towardtumor antigens with a ligand that results in oligomerization. The ligandis a protein or a non-protein. More particularly, the ligand is anon-protein, for example, a dimeric FK506 and/or dimeric FK506 analogs.The immune response is positively regulated by dimeric FK506 and/ordimeric FK506 analogs or is negatively regulated by monomeric FK506and/or monomeric FK506 analogs. More specifically, the transduced,loaded antigen-presenting cells are administered to the subjectintradermally, subcutaneously, intranodally or intralymphatically. It isenvisioned that the antigen-presenting cells are transduced with theexpression vector in vitro or ex vivo prior to administering to thesubject.

Loading the antigen-presenting cells with an antigen can be accomplishedutilizing standard methods, for example, pulsing, transducing,transfecting, and/or electrofusing. It is envisioned that the antigencan be nucleic acids (DNA or RNA), proteins, protein lysate, whole celllysate, or antigen proteins linked to other proteins, i.e., heat shockproteins.

The antigens can be derived or isolated from a pathogenic microorganismsuch as viruses including HIV, influenza, Herpes simplex, humanpapilloma virus, Hepatitis B, Hepatitis C, EBV, Cytomegalovirus (CMV)and the like. The antigen may be derived or isolated from pathogenicbacteria such as from Chlamydia, Mycobacteria, Legionella,Meningiococcus, Group A Streptococcus, Salmonella, Listeria, Hemophilusinfluenzae, and the like. Still further, the antigen may be derived orisolated from pathogenic yeast including Aspergillus, invasive Candida,Nocardia, Histoplasmosis, Cryptosporidia and the like. The antigen maybe derived or isolated from a pathogenic protozoan and pathogenicparasites including, but not limited to Pneumocystis carinii,Trypanosoma, Leishmania, Plasmodium and Toxoplasma gondii.

In certain embodiments, the antigen includes an antigen associated witha preneoplastic or hyperplastic state. Antigens may also be associatedwith, or causative of cancer. Such antigens are tumor specific antigen,tumor associated antigen (TAA) or tissue specific antigen, epitopethereof, and epitope agonist thereof. Such antigens include but are notlimited to carcinoembryonic antigen (CEA) and epitopes thereof such asCAP-1, CAP-1-6D (46) and the like, MART-1, MAGE-1, MAGE-3, GAGE, GP-100,MUC-1, MUC-2, point mutated ras oncogene, normal and point mutated p53oncogenes, PSMA, tyrosinase, TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA,CA-125, PSA, HER-2/neu/c-erb/B2, BRC-I, BRC-II, bcr-abl, pax3-fkhr,ews-fli-1, modifications of TAAs and tissue specific antigen, splicevariants of TAAs, epitope agonists, and the like.

Another embodiment is a method of treating and/or preventing a diseaseand/or disorder comprising administering to a subject an effectiveamount of an expression vector to treat and/or prevent the diseaseand/or disorder, wherein the expression vector comprises apolynucleotide promoter sequence, a polynucleotide sequence encoding aligand-binding region, a second polynucleotide sequence encoding aligand-binding region, a polynucleotide sequence encoding amembrane-targeting sequence, and a polynucleotide sequence encoding aco-stimulatory polypeptide, all operatively linked. The co-stimulatorypolypeptide is a CD40 cytoplasmic domain.

In certain embodiments, the disease is a hyperproliferative disease,which can also be further defined as cancer. In still furtherembodiments, the cancer is melanoma, non-small cell lung, small-celllung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma,glioblastoma, gum, tongue, neuroblastoma, head, neck, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder.The cancer may include a tumor comprised of tumor cells. For example,tumor cells may include, but are not limited to melanoma cell, a bladdercancer cell, a breast cancer cell, a lung cancer cell, a colon cancercell, a prostate cancer cell, a liver cancer cell, a pancreatic cancercell, a stomach cancer cell, a testicular cancer cell, a brain cancercell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancercell, a brain cancer cell, a bone cancer cell, or a soft tissue cancercell.

In other embodiments, the hyperproliferative disease is rheumatoidarthritis, inflammatory bowel disease, osteoarthritis, leiomyomas,adenomas, lipomas, hemangiomas, fibromas, vascular occlusion,restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatoushyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ,oral hairy leukoplakia, or psoriasis.

Yet further, another embodiment is a method of treating a disease and/ordisorder comprising administering to a subject an effective amount of atransduced antigen-presenting cell to treat the disease and/or disorder,wherein the transduced antigen-presenting cell is transduced with anexpression vector comprising a polynucleotide promoter sequence, a firstpolynucleotide sequence encoding a ligand-binding region, a secondpolynucleotide sequence encoding a ligand-binding region, apolynucleotide sequence encoding a membrane-targeting sequence, and apolynucleotide sequence encoding a co-stimulatory polypeptide, alloperatively linked. The co-stimulatory polypeptide is a member of theTNF Receptor family, more specifically; the co-stimulatory polypeptideis a CD40 cytoplasmic domain. The transduced antigen-presenting cellsare administered to the subject intradermally, subcutaneously, orintranodally. The antigen-presenting cells are transduced with theexpression vector in vitro prior to administering to the subject. Themethod may further comprise electrofusing the transducedantigen-presenting cell to a tumor cell. In certain embodiments, thetumor cell is a prostate tumor cell. The tumor cell is syngeneic, orallogeneic. The method may also further comprises transfecting thetransduced antigen-presenting cell with tumor cell mRNA and/or pulsingthe transduced antigen-presenting cell with tumor cell protein lysatesand/or pulsing the transduced antigen-presenting cell with heat shockproteins linked to tumor cell polypeptides.

Another embodiment is a method of treating a subject with cancercomprising administering to the patient an effective amount of atransduced antigen-presenting cell to treat the cancer, wherein thetransduced antigen-presenting cell is transduced with an expressionvector comprising a polynucleotide promoter sequence, a firstpolynucleotide sequence encoding a ligand-binding region, a secondpolynucleotide sequence encoding a ligand-binding region, apolynucleotide sequence encoding a myristoylation-targeting sequence,and a polynucleotide sequence encoding a co-stimulatory polypeptide, alloperatively linked; and administering at least one other anticancertreatment. The anticancer treatment is selected from the groupconsisting of chemotherapy, immunotherapy, surgery, radiotherapy, genetherapy and biotherapy.

Another embodiment is a transgenic mouse having incorporated into itsgenome an expression vector comprising a polynucleotide promotersequence, a polynucleotide sequence encoding a CD40 cytoplasmic domainand a polynucleotide sequence encoding a ligand-binding region, alloperatively linked. The ligand-binding region is a FKBP binding domain.The expression vector may further comprise a second ligand-bindingregion, whish is FKBP binding domain. Still further, the vector maycomprise a polynucleotide sequence encoding a myristoylation-targetingsequence. The polynucleotide promoter sequence comprises CD11c.Embryonic stem cells and/or antigen-presenting cells may be isolatedfrom the transgenic mouse.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated that the conception and specific embodimentdisclosed may be readily utilized as a basis for modifying or designingother structures for carrying out the same purposes of the presentinvention. It should also be realized that such equivalent constructionsdo not depart from the invention as set forth in the appended claims.The novel features which are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing.

FIG. 1A-FIG. 1C show the chemically induced dimerization of CD40. FIG.1A shows a model of endogenous CD40. FIG. 1B shows Chemically InducedDimerization System (CID) utilizes a lipid-permeable organic dimerizerdrug (AP20187) that binds with high affinity to drug binding domains.FIG. 1C shows NFκB-SEAP Reporter Assay in Jurkat TAg Cells.

FIG. 2A-2F show inducible CD40 initiates a potent NFkB signal in DCs.FIG. 2A shows anti-HA Western Blot Analysis of iCD40 D2SC/1 DC Clones.FIG. 2B shows anti-HA Immunofluorescence of D2SC/1.Hi (10 and D2SC/1(rt) FIG. 2C shows NFkB-SEAP Reporter Assay. FIG. 2D shows induction ofRelB and Sp1 in D2SC/1.Hi by AP20187. FIG. 2E shows maximumconcentrations of each agent (based on titrations) were co-incubatedwith iCD40 D2SC/1 for 24 hrs and nuclear lysates were analyzed bywestern blot. FIG. 2F shows kinetics of RelB activation by iCD40 orother indicated treatments.

FIG. 3A-FIG. 3C show inducible CD40 triggers DC maturation andactivation. FIG. 3A show flow cytometry of activation markers on D2SC/1treated with control, AP20187, LPS, iCD40 expression, iCD40+AP20187.FIG. 3B show reduction of phagocytosis of FITC-dextran after treatmentwith LPS or iCD40. FIG. 3C show activation of bulk lymph node cells orpurified CD8 T cells by treated D2SC/1 cells.

FIG. 4A-4E show in vivo drug-mediated activation of iCD40 DCs followingvaccination induces an enhanced antigen-specific T cell response. FIG.4A show schema of iCD40.D2SC/1-based vaccines. FIG. 4B show iCD40.D2SC/1cells were prepared for injection by in vitro LPS or iCD40 treatment, byin vivo iCD40 signaling, or by both in vitro and in vivo iCD40signaling. After 10 days, splenocytes were isolated and assayed forantigen-specific proliferation. FIG. 4C show percent ofK^(d)LLO-specific T cells from vaccinated or control mice was calculatedusing tetramer staining. FIG. 4D show CTL activity from splenocytes ofmice vaccinated with β-gal pulsed DCs treated as above using standard5-day assay using β-gal expressing target cells. FIG. 4E show CTLactivity assayed on LLO-expressing tumor cells (construct shown).

FIG. 5A-5E show iCD40 activates primary DCs and prolongs theirlongevity. FIG. 5A show Western blot (α-HA) of primary DCs infected withAD-iCD40-GFP. FIG. 5B show flow cytometry analysis of transduced DCs.FIG. 5C show flow cytometry of K^(b), B7.2 and endogenous CD40 oniCD40-stimulated DCs. FIG. 5D show kinetics of IL-12 induction (ELISA)by iCD40 and LPS. FIG. 5E show survival kinetics of DCs following CD40Lor iCD40 stimulation.

FIG. 6A-6B show iCD40 augments the immunogenicity of DNA vaccines invivo. FIG. 6A and FIG. 6C shows co-injection of an iCD40-expressingplasmid enhances antigen-specific CD8+ T cell Responses. iCD40 wassubcloned into a PCMV-driven bicistronic vector co-expressing hrGFP.Gold micro-particles were coated with plasmid DNA encoding the SIINFEKLminigene, the iCD40-hrGFP construct, or both. DNA micro-particles wereinjected into mice in the abdomen (2×) and in each ear using a heliumgene gun. DNA doses were kept constant at 2.5 μg per shot or 10 μg permouse. AP20187 was injected i.p. 20 hours later into some groups.Spleens were harvested 12 days later and analyzed by two color flowanalysis using PE-KbSIINFEKL tetramer/FITC-anti-CD8 staining. FIG. 6Bshows in vivo drug delivery enhances CD8+ T cell Activation. Splenocytesharvested above were co-incubated with 10 μg/mL SIINFEKL peptideovernight and analyzed for dual CD8+CD69+ surface expression by flowcytometry. Only viable cells were gated.

FIG. 7A-FIG. 7B show iCD40 enhancement of DNA vaccination. FIG. 7A showsthe activation of CD8+ T cells and FIG. 7B shows the activation of(CD69+) CD8+ T cells following vaccination.

FIG. 8A-FIG. 8C show CD40L downregulates and reduces the signalingcapacity of CD40. FIG. 8A shows endocytosis inhibition reduces CD40downregulation. D2SC/1 cell lines were incubated with 25011.Mcytochalasin B for 1 hour followed by a 30 min CD40L treatment. D2SC/1cells were also treated with cytochalasin B, the DMSO solvent control,and CD40L alone. Ktdepletion of the D2SC/1 cell line was also carriedout prior to CD40 surface staining and flow cytometry analysis. Onlyviable cells were gated for analysis. FIG. 8B shows inhibition oflysosomal degradation enhances intracellular CD40 levels. D2SC/1 celllines were incubated with 0.5 μM bafilomycin A inhibitor for 1 hourfollowed by intracellular staining for CD40 (Total CD40). Total CD40 iscompared to surface CD40 fluorescence. FIG. 8C shows inhibition ofendocytosis intensifies the CD40 activation signal in DC Lines. Stainingand analysis of surface H-2K^(d).

FIG. 9A-FIG. 9D show iCD40 circumvents negative feedback inhibition bythe Type II CD40 (IICD40) isoform. FIG. 9A shows a schematic of Type I,II, and iCD40. FIG. 9B shows IICD40-expressing DC lines do not expressreduced levels of iCD40. The type II CD40 isoform was rt-PCR amplifiedfrom purified BMDCs, subcloned into a pEF-1 α-driven myc-tagged ZeoRvector, and transfected into iCD40-expressing D2SC/1 cells. Doubleclonal stable lines were generated by G418/zeocin selection and limitingdilution. Resulting lines were screened for IICD40 expression byanti-myc western blot and analyzed for iCD40 expression by anti-HAwestern blots. FIG. 9C shows that Type II CD40 down-regulates surfaceexpression of Type I CD40 in DC Lines. Empty vector control andIICD40-expressing D2SC/1 lines were analyzed for their surfaceexpression of CD40 by flow cytometry. Only viable cells were gated foranalysis. FIG. 9D shows the Type II CD40 isoform downmodulates Type ICD40 signaling, but not iCD40 signaling. iCD40-IICD40-expressing D2SC/1cell lines were cultured in the presence of increasing concentrations ofCD40L and the AP20187 drug followed by surface staining and flowanalysis of H-2K^(d).

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

I. DEFINITIONS

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having”, “including”, “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “allogeneic” as used herein, refers to cell types or tissuesthat are antigenically distinct. Thus, cells or tissue transferred fromthe same species can be antigenically distinct.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.Exemplary organisms include but are not limited to, Helicobacters,Campylobacters, Clostridia, Corynebacterium diphtheriae, Bordetellapertussis, influenza virus, parainfluenza viruses, respiratory syncytialvirus, Borrelia burgdorfei, Plasmodium, herpes simplex viruses, humanimmunodeficiency virus, papillomavirus, Vibrio cholera, E. coli, measlesvirus, rotavirus, shigella, Salmonella typhi, Neisseria gonorrhea.Therefore, a skilled artisan realizes that any macromolecule, includingvirtually all proteins or peptides, can serve as antigens. Furthermore,antigens can be derived from recombinant or genomic DNA. A skilledartisan realizes that any DNA, which contains nucleotide sequences orpartial nucleotide sequences of a pathogenic genome or a gene or afragment of a gene for a protein that elicits an immune response resultsin synthesis of an antigen. Furthermore, one skilled in the art realizesthat the present invention is not limited to the use of the entirenucleic acid sequence of a gene or genome. It is readily inherent thatthe present invention includes, but is not limited to, the use ofpartial nucleic acid sequences of more than one gene or genome and thatthese nucleic acid sequences are arranged in various combinations toelicit the desired immune response.

The term “antigen-presenting cell” is any of a variety of cells capableof displaying, acquiring, or presenting at least one antigen orantigenic fragment on (or at) its cell surface. In general, the term“antigen-presenting cell” can be any cell that accomplishes the goal ofthe invention by aiding the enhancement of an immune response (i.e.,from the T-cell or -B-cell arms of the immune system) against an antigenor antigenic composition. Such cells can be defined by those of skill inthe art, using methods disclosed herein and in the art. As is understoodby one of ordinary skill in the art (see for example Kuby, 1993,incorporated herein by reference), and used herein certain embodiments,a cell that displays or presents an antigen normally or preferentiallywith a class II major histocompatibility molecule or complex to animmune cell is an “antigen-presenting cell.” In certain aspects, a cell(e.g., an APC cell) may be fused with another cell, such as arecombinant cell or a tumor cell that expresses the desired antigen.Methods for preparing a fusion of two or more cells is well known in theart, such as for example, the methods disclosed in Goding, pp. 65-66,71-74 1986; Campbell, pp. 75-83, 1984; Kohler and Milstein, 1975; Kohlerand Milstein, 1976, Gefter et al., 1977, each incorporated herein byreference. In some cases, the immune cell to which an antigen-presentingcell displays or presents an antigen to is a CD4+ TH cell. Additionalmolecules expressed on the APC or other immune cells may aid or improvethe enhancement of an immune response. Secreted or soluble molecules,such as for example, cytokines and adjuvants, may also aid or enhancethe immune response against an antigen. Such molecules are well known toone of skill in the art, and various examples are described herein.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma,astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck,breast, pancreatic, prostate, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,sarcoma or bladder.

The terms “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

As used herein, the term “iCD40 molecule” is defined as an inducibleCD40. This iCD40 can bypass mechanisms that extinguish endogenous CD40signaling. The term “iCD40” embraces “iCD40 nucleic acids”, “iCD40polypeptides” and/or iCD40 expression vectors. Yet further, it isunderstood the activity of iCD40 as used herein is driven by CID.

As used herein, the term “cDNA” is intended to refer to DNA preparedusing messenger RNA (mRNA) as template. The advantage of using a cDNA,as opposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There are times when thefull or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

The term “dendritic cell” (DC) is an antigen presenting cell existing invivo, in vitro, ex vivo, or in a host or subject, or which can bederived from a hematopoietic stem cell or a monocyte. Dendritic cellsand their precursors can be isolated from a variety of lymphoid organs,e.g., spleen, lymph nodes, as well as from bone marrow and peripheralblood. The DC has a characteristic morphology with thin sheets(lamellipodia) extending in multiple directions away from the dendriticcell body. Typically, dendritic cells express high levels of MHC andcostimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells caninduce antigen specific differentiation of T cells in vitro, and areable to initiate primary T cell responses in vitro and in vivo.

As used herein, the term “expression construct” or “transgene” isdefined as any type of genetic construct containing a nucleic acidcoding for gene products in which part or all of the nucleic acidencoding sequence is capable of being transcribed can be inserted intothe vector. The transcript is translated into a protein, but it need notbe. In certain embodiments, expression includes both transcription of agene and translation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encodinggenes of interest. In the present invention, the term “therapeuticconstruct” may also be used to refer to the expression construct ortransgene. One skilled in the art realizes that the present inventionutilizes the expression construct or transgene as a therapy to treathyperproliferative diseases or disorders, such as cancer, thus theexpression construct or transgene is a therapeutic construct or aprophylactic construct.

As used herein, the term “expression vector” refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra.

As used herein, the term “ex vivo” refers to “outside” the body. One ofskill in the art is aware that ex vivo and in vitro can be usedinterchangeably.

As used herein, the term “functionally equivalent”, refers to CD40nucleic acid fragment, variant, or analog, refers to a nucleic acid thatcodes for a CD40 polypeptide that stimulates an immune response todestroy tumors or hyperproliferative disease. Preferably “functionallyequivalent” refers to an CD40 polypeptide that is lacking theextracellular domain, but is capable of amplifying the T cell-mediatedtumor killing response by upregulating dendritic cell expression ofantigen presentation molecules.

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occulsion,restenosis, atherosclerosis, or inflammatory bowel disease.

As used herein, the term “gene” is defined as a functional protein,polypeptide, or peptide-encoding unit. As will be understood by those inthe art, this functional term includes genomic sequences, cDNAsequences, and smaller engineered gene segments that express, or isadapted to express, proteins, polypeptides, domains, peptides, fusionproteins, and mutants.

The term “immunogenic composition” or “immunogen” refers to a substancethat is capable of provoking an immune response. Examples of immunogensinclude, e.g., antigens, autoantigens that play a role in induction ofautoimmune diseases, and tumor-associated antigens expressed on cancercells.

The term “immunocompromised” as used herein is defined as a subject thathas reduced or weakened immune system. The immunocompromised conditionmay be due to a defect or dysfunction of the immune system or to otherfactors that heighten susceptibility to infection and/or disease.Although such a categorization allows a conceptual basis for evaluation,immunocompromised individuals often do not fit completely into one groupor the other. More than one defect in the body's defense mechanisms maybe affected. For example, individuals with a specific T-lymphocytedefect caused by HIV may also have neutropenia caused by drugs used forantiviral therapy or be immunocompromised because of a breach of theintegrity of the skin and mucous membranes. An immunocompromised statecan result from indwelling central lines or other types of impairmentdue to intravenous drug abuse; or be caused by secondary malignancy,malnutrition, or having been infected with other infectious agents suchas tuberculosis or sexually transmitted diseases, e.g., syphilis orhepatitis.

As used herein, the term “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

As used herein, the term “polynucleotide” is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means. Furthermore, one skilled in the art iscognizant that polynucleotides include mutations of the polynucleotides,include but are not limited to, mutation of the nucleotides, ornucleosides by methods well known in the art.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is interchangeable with the terms “peptides” and“proteins”.

As used herein, the term “promoter” is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene.

As used herein, the term “regulate an immune response” or “modulate animmune response” refers to the ability to modify the immune response.For example, the composition of the present invention is capable ofenhancing and/or activating the immune response. Still further, thecomposition of the present invention is also capable of inhibiting theimmune response. The form of regulation is determined by the ligand thatis used with the composition of the present invention. For example, adimeric analog of the chemical results in dimerization of theco-stimulatory polypeptide leading to activation of the DCs, however, amonomeric analog of the chemical does not result in dimerization of theco-stimulatory polypeptide, which would not activate the DCs.

The term “transfection” and “transduction” are interchangeable and referto the process by which an exogenous DNA sequence is introduced into aeukaryotic host cell. Transfection (or transduction) can be achieved byany one of a number of means including electroporation, microinjection,gene gun delivery, retroviral infection, lipofection, superfection andthe like.

As used herein, the term “syngeneic” refers to cells, tissues or animalsthat have genotypes. For example, identical twins or animals of the sameinbred strain. Syngeneic and isogeneic can be used interchangeable.

The term “subject” as used herein includes, but is not limited to, anorganism or animal; a mammal, including, e.g., a human, non-humanprimate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig,hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal,including, e.g., a non-mammalian vertebrate, such as a bird (e.g., achicken or duck) or a fish, and a non-mammalian invertebrate.

As used herein, the term “under transcriptional control” or “operativelylinked” is defined as the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

As used herein, the terms “treatment”, “treat”, “treated”, or “treating”refer to prophylaxis and/or therapy. When used with respect to aninfectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a formthat is capable of being administered to an animal. Typically, thevaccine comprises a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, the composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a subject, the vaccine is able toprovoke an immune response including, but not limited to, the productionof antibodies, cytokines and/or other cellular responses.

II. DENDRITIC CELLS

The innate immune system uses a set of germline-encoded receptors forthe recognition of conserved molecular patterns present inmicroorganisms. These molecular patterns occur in certain constituentsof microorganisms including: lipopolysaccharides, peptidoglycans,lipoteichoic acids, phosphatidyl cholines, bacteria-specific proteins,including lipoproteins, bacterial DNAs, viral single and double-strandedRNAs, unmethylated CpG-DNAs, mannans and a variety of other bacterialand fungal cell wall components. Such molecular patterns can also occurin other molecules such as plant alkaloids. These targets of innateimmune recognition are called Pathogen Associated Molecular Patterns(PAMPs) since they are produced by microorganisms and not by theinfected host organism (Janeway et al., 1989; Medzhitov et al., 1997).

The receptors of the innate immune system that recognize PAMPs arecalled Pattern Recognition Receptors (PRRs) (Janeway et al., 1989;Medzhitov et al., 1997). These receptors vary in structure and belong toseveral different protein families. Some of these receptors recognizePAMPs directly (e.g., CD14, DEC205, collectins), while others (e.g.,complement receptors) recognize the products generated by PAMPrecognition. Members of these receptor families can, generally, bedivided into three types: 1) humoral receptors circulating in theplasma; 2) endocytic receptors expressed on immune-cell surfaces, and 3)signaling receptors that can be expressed either on the cell surface orintracellularly (Medzhitov et al., 1997; Fearon et al., 1996).

Cellular PRRs are expressed on effector cells of the innate immunesystem, including cells that function as professional antigen-presentingcells (APC) in adaptive immunity. Such effector cells include, but arenot limited to, macrophages, dendritic cells, B lymphocytes and surfaceepithelia. This expression profile allows PRRs to directly induce innateeffector mechanisms, and also to alert the host organism to the presenceof infectious agents by inducing the expression of a set of endogenoussignals, such as inflammatory cytokines and chemokines, as discussedbelow. This latter function allows efficient mobilization of effectorforces to combat the invaders.

The primary function of dendritic cells (DCs) is to acquire antigen inthe peripheral tissues, travel to secondary lymphoid tissue, and presentantigen to effector T cells of the immune system (Banchereau, et al.,2000; Banchereau, et al., 1998). As DCs carry out their crucial role inthe immune response, they undergo maturational changes allowing them toperform the appropriate function for each environment (Termeer, C. C. etal., 2000). During DC maturation, antigen uptake potential is lost, thesurface density of major histocompatibility complex (MHC) class I andclass II molecules increases by 10-100 fold, and CD40, costimulatory andadhesion molecule expression also greatly increases (Lanzavecchia, A. etal., 2000). In addition, other genetic alterations permit the DCs tohome to the T cell-rich paracortex of draining lymph nodes and toexpress T-cell chemokines that attract naive and memory T cells andprime antigen-specific naive THO cells (Adema, G. J. et al., 1997).During this stage, mature DCs present antigen via their MHC II moleculesto CD4+ T helper cells, inducing the upregulation of T cell CD40 ligand(CD40L) that, in turn, engages the DC CD40 receptor. This DC:T cellinteraction induces rapid expression of additional DC molecules that arecrucial for the initiation of a potent CD8+ cytotoxic T lymphocyte (CTL)response, including further upregulation of MHC I and II molecules,adhesion molecules, costimulatory molecules (e.g., B7.1,B7.2), cytokines(e.g., IL-12) and anti-apoptotic proteins (e.g., Bcl-2) (Anderson, D.M., et al., 1997; Caux, C., et al., 1997; Ohshima, Y., et al., 1997;Sallusto, F., et al., 1998). CD8+ T cells exit lymph nodes, reentercirculation and home to the original site of inflammation to destroypathogens or malignant cells.

One key parameter influencing the function of DCs is the CD40 receptor,serving as the “on switch” for DCs (Bennett, S. R. et al., 1998; Clark,S. R. et al., 2000; Fernandez, N. C., et al., 1999; Ridge, J. P. et al.,1998; Schoenberger, S. P., et al., 1998). CD40 is a 48-kDa transmembranemember of the TNF receptor superfamily (McWhirter, S. M., et al., 1999).CD40-CD40L interaction induces CD40 trimerization, necessary forinitiating signaling cascades involving TNF receptor associated factors(TRAFs) (Ni, C. Z., et al., 2000; Pullen, S. S. et al., 1999). CD40 usesthese signaling molecules to activate several transcription factors inDCs, including NFκB, AP-1, STATS, and p38MAPK (McWhirter, S. M., et al.,1999).

The present invention contemplates a novel DC activation system based onrecruiting signaling molecules or co-stimulatory polypeptides to theplasmid membrane of the DCs resulting in prolonged/increased activationand/or survival in the DCs. Co-stimulatory polypeptides include anymolecule or polypeptide that activates the NFκB pathway, Akt pathway,and/or p38 pathway. The DC activation system is based upon utilizing arecombinant signaling molecule fused to a ligand-binding domains (i.e.,a small molecule binding domain) in which the co-stimulatory polypeptideis activated and/or regulated with a ligand resulting in oligomerization(i.e., a lipid-permeable, organic, dimerizing drug). Other systems thatmay be used to crosslink or oligomerization of co-stimulatorypolypeptides include antibodies, natural ligands, and/or artificialcross-reacting or synthetic ligands. Yet further, other dimerizationsystems contemplated include the coumermycin/DNA gyrase B system.

Co-stimulatory polypeptides that can be used in the present inventioninclude those that activate NFκB and other variable signaling cascadesfor example the p38 pathway and/or Akt pathway. Such co-stimulatorypolypeptides include, but are not limited to Pattern RecognitionReceptors, C-reactive protein receptors (i.e., Nod1, Nod2, PtX3-R), TNFreceptors (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), and HSP receptors(Lox-1 and CD-91).

Pattern Recognition Receptors include, but are not limited to endocyticpattern-recognition receptors (i.e., mannose receptors, scavengerreceptors (i.e., Mac-1, LRP, peptidoglycan, techoic acids, toxins,CD11c/CR4)); external signal pattern-recognition receptors (Toll-likereceptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10),peptidoglycan recognition protein, (PGRPs bind bacterial peptidoglycan,and CD14); and internal signal pattern-recognition receptors (i.e.,NOD-receptors 1 & 2).

III. ENGINEERING EXPRESSION CONSTRUCTS

The present invention involves an expression construct encoding aco-stimulatory polypeptide and a ligand-binding domain, all operativelylinked. More particularly, more than one ligand-binding domain is usedin the expression construct. Yet further, the expression constructcontains a membrane-targeting sequence. One with skill in the artrealizes that appropriate expression constructs may include aco-stimulatory polypeptide element on either side of the above FKBPligand-binding elements. The expression construct of the presentinvention may be inserted into a vector, for example a viral vector orplasmid.

A. Co-Stimulatory Polypeptides

In the present invention, co-stimulatory polypeptide molecules arecapable of amplifying the T-cell-mediate response by upregulatingdendritic cell expression of antigen presentation molecules.Co-stimulatory proteins that are contemplated in the present inventioninclude, for example, but are not limited to the members of tumornecrosis factor (TNF) family (i.e., CD40, RANK/TRANCE-R, OX40, 4-1B),Toll-like receptors, C-reactive protein receptors, Pattern RecognitionReceptors, and HSP receptors. Typically, the cytoplasmic domains fromthese co-stimulatory polypeptides are used in the expression vector. Thecytoplasmic domain from one of the various co-stimulatory polypeptides,including mutants thereof, where the recognition sequence involved ininitiating transcription associated with the cytoplasmic domain is knownor a gene responsive to such sequence is known.

In specific embodiments of the present invention, the co-stimulatorypolypeptide molecule is CD40. The CD40 molecule comprises a nucleic acidmolecule which: (1) hybridizes under stringent conditions to a nucleicacid having the sequence of a known CD40 gene and (2) codes for an CD40polypeptide. Preferably the CD40 polypeptide is lacking theextracellular domain. It is contemplated that other normal or mutantvariants of CD40 can be used in the present invention. Exemplarypolynucleotide sequences that encode CD40 polypeptides include, but arenot limited to SEQ.ID.NO: 1 and CD40 isoforms from other species.

In certain embodiments, the present invention involves the manipulationof genetic material to produce expression constructs that encode aninducible form of CD40 (iCD40). Such methods involve the generation ofexpression constructs containing, for example, a heterologous nucleicacid sequence encoding CD40 cytoplasmic domain and a means for itsexpression, replicating the vector in an appropriate helper cell,obtaining viral particles produced therefrom, and infecting cells withthe recombinant virus particles.

Thus, the preferable CD40 molecule of the present invention lacks theextracellular domain. In specific embodiments, the extracellular domainis truncated or removed. It is also contemplated that the extracellulardomain can be mutated using standard mutagenesis, insertions, deletions,or substitutions to produce an CD40 molecule that does not have afunctional extracellular domain. The preferred CD40 nucleic acid has thenucleic acid sequence of SEQ.ID.NO. 2. The CD40 nucleic acids of theinvention also include homologs and alleles of a nucleic acid having thesequence of SEQ.ID.NO. 2, as well as, functionally equivalent fragments,variants, and analogs of the foregoing nucleic acids.

In the context of gene therapy, the gene will be a heterologouspolynucleotide sequence derived from a source other than the viralgenome, which provides the backbone of the vector. The gene is derivedfrom a prokaryotic or eukaryotic source such as a bacterium, a virus,yeast, a parasite, a plant, or even an animal. The heterologous DNA alsois derived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA also may include a regulatorysequence, which is derived from one source and the gene from a differentsource.

B. Ligand-Binding Domains

The ligand-binding (“dimerization”) domain of the expression constructof this invention can be any convenient domain that will allow forinduction using a natural or unnatural ligand, preferably an unnaturalsynthetic ligand. The ligand-binding domain can be internal or externalto the cellular membrane, depending upon the nature of the construct andthe choice of ligand. A wide variety of ligand-binding proteins,including receptors, are known, including ligand-binding proteinsassociated with the cytoplasmic regions indicated above. As used hereinthe term “ligand-binding domain can be interchangeable with the term“receptor”. Of particular interest are ligand-binding proteins for whichligands (preferably small organic ligands) are known or may be readilyproduced. These ligand-binding domains or receptors include the FKBPsand cyclophilin receptors, the steroid receptors, the tetracyclinereceptor, the other receptors indicated above, and the like, as well as“unnatural” receptors, which can be obtained from antibodies,particularly the heavy or light chain subunit, mutated sequencesthereof, random amino acid sequences obtained by stochastic procedures,combinatorial syntheses, and the like.

For the most part, the ligand-binding domains or receptor domains willbe at least about 50 amino acids, and fewer than about 350 amino acids,usually fewer than 200 amino acids, either as the natural domain ortruncated active portion thereof. Preferably the binding domain will besmall (<25 kDa, to allow efficient transfection in viral vectors),monomeric (this rules out the avidin-biotin system), nonimmunogenic, andshould have synthetically accessible, cell permeable, nontoxic ligandsthat can be configured for dimerization.

The receptor domain can be intracellular or extracellular depending uponthe design of the expression construct and the availability of anappropriate ligand. For hydrophobic ligands, the binding domain can beon either side of the membrane, but for hydrophilic ligands,particularly protein ligands, the binding domain will usually beexternal to the cell membrane, unless there is a transport system forinternalizing the ligand in a form in which it is available for binding.For an intracellular receptor, the construct can encode a signal peptideand transmembrane domain 5′ or 3′ of the receptor domain sequence or byhaving a lipid attachment signal sequence 5′ of the receptor domainsequence. Where the receptor domain is between the signal peptide andthe transmembrane domain, the receptor domain will be extracellular.

The portion of the expression construct encoding the receptor can besubjected to mutagenesis for a variety of reasons. The mutagenizedprotein can provide for higher binding affinity, allow fordiscrimination by the ligand of the naturally occurring receptor and themutagenized receptor, provide opportunities to design a receptor-ligandpair, or the like. The change in the receptor can involve changes inamino acids known to be at the binding site, random mutagenesis usingcombinatorial techniques, where the codons for the amino acidsassociated with the binding site or other amino acids associated withconformational changes can be subject to mutagenesis by changing thecodon(s) for the particular amino acid, either with known changes orrandomly, expressing the resulting proteins in an appropriateprokaryotic host and then screening the resulting proteins for binding.

Antibodies and antibody subunits, e.g., heavy or light chain,particularly fragments, more particularly all or part of the variableregion, or fusions of heavy and light chain to create high-affinitybinding, can be used as the binding domain. Antibodies that arecontemplated in the present invention include ones that are anectopically expressed human product, such as an extracellular domainthat would not trigger an immune response and generally not expressed inthe periphery (i.e., outside the CNS/brain area). Such examples,include, but are not limited to low affinity nerve growth factorreceptor (LNGFR), and embryonic surface proteins (i.e., carcinoembryonicantigen).

Yet further, antibodies can be prepared against haptenic molecules,which are physiologically acceptable, and the individual antibodysubunits screened for binding affinity. The cDNA encoding the subunitscan be isolated and modified by deletion of the constant region,portions of the variable region, mutagenesis of the variable region, orthe like, to obtain a binding protein domain that has the appropriateaffinity for the ligand. In this way, almost any physiologicallyacceptable haptenic compound can be employed as the ligand or to providean epitope for the ligand. Instead of antibody units, natural receptorscan be employed, where the binding domain is known and there is a usefulligand for binding.

C. Oligomerization

The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e., as a result ofoligomerization following ligand-binding, although other binding events,for example allosteric activation, can be employed to initiate a signal.The construct of the chimeric protein will vary as to the order of thevarious domains and the number of repeats of an individual domain.

For multimerizing the receptor, the ligand for the ligand-bindingdomains/receptor domains of the chimeric surface membrane proteins willusually be multimeric in the sense that it will have at least twobinding sites, with each of the binding sites capable of binding to thereceptor domain. Desirably, the subject ligands will be a dimer orhigher order oligomer, usually not greater than about tetrameric, ofsmall synthetic organic molecules, the individual molecules typicallybeing at least about 150 D and fewer than about 5 kDa, usually fewerthan about 3 kDa. A variety of pairs of synthetic ligands and receptorscan be employed. For example, in embodiments involving naturalreceptors, dimeric FK506 can be used with an FKBP receptor, dimerizedcyclosporin A can be used with the cyclophilin receptor, dimerizedestrogen with an estrogen receptor, dimerized glucocorticoids with aglucocorticoid receptor, dimerized tetracycline with the tetracyclinereceptor, dimerized vitamin D with the vitamin D receptor, and the like.Alternatively higher orders of the ligands, e.g., trimeric can be used.For embodiments involving unnatural receptors, e.g., antibody subunits,modified antibody subunits or modified receptors and the like, any of alarge variety of compounds can be used. A significant characteristic ofthese ligand units is that they bind the receptor with high affinity andare able to be dimerized chemically.

In certain embodiments, the present invention utilizes the technique ofchemically induced dimerization (CID) to produce a conditionallycontrolled protein or polypeptide. In addition to this technique beinginducible, it also is reversible, due to the degradation of the labiledimerizing agent or administration of a monomeric competitive inhibitor.

CID system uses synthetic bivalent ligands to rapidly crosslinksignaling molecules that are fused to ligand-binding domains CID. Thissystem has been used to trigger the oligomerization and activation ofcell surface (Spencer et al., 1993; Spencer et al., 1996; Blau et al.,1997), or cytosolic proteins (Luo et al., 1996; MacCorkle et al., 1998),the recruitment of transcription factors to DNA elements to modulatetranscription (Ho et al., 1996; Rivera et al., 1996) or the recruitmentof signaling molecules to the plasma membrane to stimulate signaling(Spencer et al., 1995; Holsinger et al., 1995).

The CID system is based upon the notion that surface receptoraggregation effectively activates downstream signaling cascades. In thesimplest embodiment, the CID system uses a dimeric analog of the lipidpermeable immunosuppressant drug, FK506, which loses its normalbioactivity while gaining the ability to crosslink molecules geneticallyfused to the FK506-binding protein, FKBP12. By fusing one or more FKBPsand a myristoylation sequence to the cytoplasmic signaling domain of atarget receptor, one can stimulate signaling in a dimerizerdrug-dependent, but ligand and ectodomain-independent manner. Thisprovides the system with temporal control, reversibility using monomericdrug analogs, and enhanced specificity. The high affinity ofthird-generation AP20187/AP1903 CIDs for their binding domain, FKBP12permits specific activation of the recombinant receptor in vivo withoutthe induction of non-specific side effects through endogenous FKBP12. Inaddition, the synthetic ligands are resistant to protease degradation,making them more efficient at activating receptors in vivo than mostdelivered protein agents.

The ligands used in the present invention are capable of binding to twoor more of the ligand-binding domains. One skilled in the art realizesthat the chimeric proteins may be able to bind to more than one ligandwhen they contain more than one ligand-binding domain. The ligand istypically a non-protein or a chemical. Exemplary ligands include, butare not limited to dimeric FK506 (e.g., FK1012).

Since the mechanism of CD40 activation is fundamentally based ontrimerization, this receptor is particularly amenable to the CID system.CID regulation provides the system with 1) temporal control, 2)reversibility by addition of a non-active monomer upon signs of anautoimmune reaction, and 3) limited potential for non-specific sideeffects. In addition, inducible in vivo DC CD40 activation wouldcircumvent the requirement of a second “danger” signal normally requiredfor complete induction of CD40 signaling and would potentially promoteDC survival in situ allowing for enhanced T cell priming Thus,engineering DC vaccines to express iCD40 amplifies the T cell-mediatedkilling response by upregulating DC expression of antigen presentationmolecules, adhesion molecules, TH1 promoting cytokines, and pro-survivalfactors.

Other dimerization systems contemplated include the coumermycin/DNAgyrase B system. Coumermycin-induced dimerization activates a modifiedRaf protein and stimulates the MAP kinase cascade. See Farrar et al.,1996.

D. Membrane-Targeting

A membrane-targeting sequence provides for transport of the chimericprotein to the cell surface membrane, where the same or other sequencescan encode binding of the chimeric protein to the cell surface membrane.Any membrane-targeting sequence can be employed that is functional inthe host and may, or may not, be associated with one of the otherdomains of the chimeric protein. Such sequences include, but are notlimited to myristoylation-targeting sequence, palmitoylation targetingsequence, prenylation sequences (i.e., farnesylation,geranyl-geranylation, CAAX Box) or transmembrane sequences (utilizingsignal peptides) from receptors.

E. Selectable Markers

In certain embodiments of the invention, the expression constructs ofthe present invention contain nucleic acid constructs whose expressionis identified in vitro or in vivo by including a marker in theexpression construct. Such markers would confer an identifiable changeto the cell permitting easy identification of cells containing theexpression construct. Usually the inclusion of a drug selection markeraids in cloning and in the selection of transformants. For example,genes that confer resistance to neomycin, puromycin, hygromycin, DHFR,GPT, zeocin and histidinol are useful selectable markers. Alternatively,enzymes such as herpes simplex virus thymidine kinase (tk) are employedImmunologic markers also can be employed. The selectable marker employedis not believed to be important, so long as it is capable of beingexpressed simultaneously with the nucleic acid encoding a gene product.Further examples of selectable markers are well known to one of skill inthe art and include reporters such as EGFP, βgal or chloramphenicolacetyltransferase (CAT).

F. Control Regions

1. Promoters

The particular promoter employed to control the expression of apolynucleotide sequence of interest is not believed to be important, solong as it is capable of directing the expression of the polynucleotidein the targeted cell. Thus, where a human cell is targeted, it ispreferable to position the polynucleotide sequence-coding regionadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally speaking, such a promoter mightinclude either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it is desirable to prohibit orreduce expression of one or more of the transgenes. Examples oftransgenes that are toxic to the producer cell line are pro-apoptoticand cytokine genes. Several inducible promoter systems are available forproduction of viral vectors where the transgene products are toxic (addin more inducible promoters).

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constitutively expressed fromone vector, whereas the ecdysone-responsive promoter, which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstitutively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constitutively on.

In some circumstances, it is desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity are utilized depending onthe level of expression desired. In mammalian cells, the CMV immediateearly promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that are used depending on the desiredeffect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoterssuch as from the E1A, E2A, or MLP region, AAV LTR, HSV-TK, and aviansarcoma virus.

Similarly tissue specific promoters are used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA associated promoter or prostate-specific glandularkallikrein.

In certain indications, it is desirable to activate transcription atspecific times after administration of the gene therapy vector. This isdone with such promoters as those that are hormone or cytokineregulatable. Cytokine and inflammatory protein responsive promoters thatcan be used include K and T kininogen (Kageyama et al., 1987), c-fos,TNF-alpha, C-reactive protein (Arcone et al., 1988), haptoglobin(Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poliand Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-1acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitrypsin,lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et al.,1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UVradiation, retinoic acid, and hydrogen peroxide), collagenase (inducedby phorbol esters and retinoic acid), metallothionein (heavy metal andglucocorticoid inducible), Stromelysin (inducible by phorbol ester,interleukin-1 and EGF), alpha-2 macroglobulin and alpha-1anti-chymotrypsin.

It is envisioned that any of the above promoters alone or in combinationwith another can be useful according to the present invention dependingon the action desired. In addition, this list of promoters should not beconstrued to be exhaustive or limiting, those of skill in the art willknow of other promoters that are used in conjunction with the promotersand methods disclosed herein.

2. Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Any promoter/enhancer combination (as per the Eukaryotic Promoter DataBase EPDB) can be used to drive expression of the gene. Eukaryotic cellscan support cytoplasmic transcription from certain bacterial promotersif the appropriate bacterial polymerase is provided, either as part ofthe delivery complex or as an additional genetic expression construct.

3. Polyadenylation Signals

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence is employed such as human or bovine growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

4. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be in-frame with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements is used to create multigene, orpolycistronic messages. IRES elements are able to bypass theribosome-scanning model of 5′ methylated cap-dependent translation andbegin translation at internal sites (Pelletier and Sonenberg, 1988).IRES elements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

IV. METHODS OF GENE TRANSFER

In order to mediate the effect of the transgene expression in a cell, itwill be necessary to transfer the expression constructs of the presentinvention into a cell. Such transfer may employ viral or non-viralmethods of gene transfer. This section provides a discussion of methodsand compositions of gene transfer.

A transformed cell comprising an expression vector is generated byintroducing into the cell the expression vector. Suitable methods forpolynucleotide delivery for transformation of an organelle, a cell, atissue or an organism for use with the current invention includevirtually any method by which a polynucleotide (e.g., DNA) can beintroduced into an organelle, a cell, a tissue or an organism, asdescribed herein or as would be known to one of ordinary skill in theart.

A host cell can, and has been, used as a recipient for vectors. Hostcells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded polynucleotide sequences. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained through the American Type Culture Collection (ATCC),which is an organization that serves as an archive for living culturesand genetic materials. In specific embodiments, the host cell is adendritic cell, which is an antigen-presenting cell.

It is well within the knowledge and skill of a skilled artisan todetermine an appropriate host. Generally this is based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5a, JM109, and KCB, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE®, La Jolla, Calif.). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses. Eukaryotic cells that can be used as host cells include,but are not limited to yeast, insects and mammals. Examples of mammalianeukaryotic host cells for replication and/or expression of a vectorinclude, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO,Saos, and PC12. Examples of yeast strains include, but are not limitedto, YPH499, YPH500 and YPH501.

A. Non-Viral Transfer

1. Ex Vivo Transformation

Methods for transfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example, canine endothelial cells have been genetically altered byretroviral gene transfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, Yucatan minipig endothelial cellswere transfected by retrovirus in vitro and transplanted into an arteryusing a double-balloon catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and transfected exvivo using the polynucleotides of the present invention. In particularaspects, the transplanted cells or tissues may be placed into anorganism. Thus, it is well within the knowledge of one skilled in theart to isolate dendritic cells from an animal, transfect the cells withthe expression vector and then administer the transfected or transformedcells back to the animal.

2. Injection

In certain embodiments, a polynucleotide may be delivered to anorganelle, a cell, a tissue or an organism via one or more injections(i.e., a needle injection), such as, for example, subcutaneously,intradermally, intramuscularly, intravenously, intraperitoneally, etc.Methods of injection of vaccines are well known to those of ordinaryskill in the art (e.g., injection of a composition comprising a salinesolution). Further embodiments of the present invention include theintroduction of a polynucleotide by direct microinjection. The amount ofthe expression vector used may vary upon the nature of the antigen aswell as the organelle, cell, tissue or organism used.

Intradermal, intranodal, or intralymphatic injections are some of themore commonly used methods of DC administration. Intradermal injectionis characterized by a low rate of absorption into the bloodstream butrapid uptake into the lymphatic system. The presence of large numbers ofLangerhans dendritic cells in the dermis will transport intact as wellas processed antigen to draining lymph nodes. Proper site preparation isnecessary to perform this correctly (i.e., hair must be clipped in orderto observe proper needle placement). Intranodal injection allows fordirect delivery of antigen to lymphoid tissues. Intralymphatic injectionallows direct administration of DCs.

3. Electroporation

In certain embodiments of the present invention, a polynucleotide isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge. In some variantsof this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference).

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

4. Calcium Phosphate

In other embodiments of the present invention, a polynucleotide isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

5. DEAE-Dextran

In another embodiment, a polynucleotide is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

6. Sonication Loading

Additional embodiments of the present invention include the introductionof a polynucleotide by direct sonic loading. LTK-fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

7. Liposome-Mediated Transfection

In a further embodiment of the invention, a polynucleotide may beentrapped in a lipid complex such as, for example, a liposome. Liposomesare vesicular structures characterized by a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is a polynucleotide complexed with Lipofectamine (GibcoBRL) or Superfect (Qiagen).

8. Receptor Mediated Transfection

Still further, a polynucleotide may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a polynucleotide-binding agent. Otherscomprise a cell receptor-specific ligand to which the polynucleotide tobe delivered has been operatively attached. Several ligands have beenused for receptor-mediated gene transfer (Wu and Wu, 1987; Wagner etal., 1990; Perales et al., 1994; Myers, EPO 0273085), which establishesthe operability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand is chosen to correspond to a receptor specificallyexpressed on the target cell population.

In other embodiments, a polynucleotide delivery vehicle component of acell-specific polynucleotide-targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The polynucleotide(s) tobe delivered are housed within the liposome and the specific bindingligand is functionally incorporated into the liposome membrane. Theliposome will thus specifically bind to the receptor(s) of a target celland deliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a polynucleotide tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the polynucleotide delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialoganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

9. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce apolynucleotide into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA-coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

In this microprojectile bombardment, one or more particles may be coatedwith at least one polynucleotide and delivered into cells by apropelling force. Several devices for accelerating small particles havebeen developed. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

B. Viral Vector-Mediated Transfer

In certain embodiments, transgene is incorporated into a viral particleto mediate gene transfer to a cell. Typically, the virus simply will beexposed to the appropriate host cell under physiologic conditions,permitting uptake of the virus. The present methods are advantageouslyemployed using a variety of viral vectors, as discussed below.

1. Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kb viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence, which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous in that elimination ofadenoviral genes serves both ends. By practice of the present invention,it is possible achieve both these goals while retaining the ability tomanipulate the therapeutic constructs with relative ease.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsulation is localizedbetween 194-385 by (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ, DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 by (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the E1A (194-358 bp)region of the genome grew poorly even in a cell line that complementedthe early (E1A) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0-353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al.,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals is packagedselectively when compared to the helpers. If the preference is greatenough, stocks approaching homogeneity should be achieved.

2. Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed, functions as a signal for packaging of the genomeinto virions. Two long terminal repeat (LTR) sequences are present atthe 5′ and 3′ ends of the viral genome. These contain strong promoterand enhancer sequences and also are required for integration in the hostcell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and Ψ components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and Ψ sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the Ψ sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. However, integration and stable expression ofmany types of retroviruses require the division of host cells (Paskindet al., 1975).

An approach designed to allow specific targeting of retrovirus vectorsrecently was developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

3. Adeno-Associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low-levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al., 1987), or by other methods knownto the skilled artisan, including but not limited to chemical orenzymatic synthesis of the terminal repeats based upon the publishedsequence of AAV. The ordinarily skilled artisan can determine, bywell-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,1995; Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al.,1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994;1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al.,1996).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1995; Flotte et al., 1993). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor IXgene delivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al.,1996; Ping et al., 1996; Xiao et al., 1996).

4. Other Viral Vectors

Other viral vectors are employed as expression constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) canarypox virus, and herpes viruses are employed. These viruses offer severalfeatures for use in gene transfer into various mammalian cells.

Once the construct has been delivered into the cell, the nucleic acidencoding the transgene are positioned and expressed at different sites.In certain embodiments, the nucleic acid encoding the transgene isstably integrated into the genome of the cell. This integration is inthe cognate location and orientation via homologous recombination (genereplacement) or it is integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid isstably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

V. ENHANCEMENT OF AN IMMUNE RESPONSE

In certain embodiments, the present invention contemplates a novel DCactivation strategy that incorporates the manipulation of signalingco-stimulatory polypeptides that activate NFκB pathways, Akt pathways,and/or p38 pathways. This DC activation system can be used inconjunction with or without standard vaccines to enhance the immuneresponse since it replaces the requirement for CD4+ T cell help duringAPC activation (Bennett S. R. et al., 1998; Ridge, J. P. et al., 1998;Schoenberger, S. P., et al., 1998). Thus, the DC activation system ofthe present invention enhances immune responses by circumventing theneed for the generation of MHC class II-specific peptides.

In specific embodiments, the DC activation is via CD40 activation. Thus,DC activation via endogenous CD40/CD40L interactions may be subject todownregulation due to negative feedback, leading rapidly to the “IL-12burn-out effect”. Within 7 to 10 hours after CD40 activation, analternatively spliced isoform of CD40 (type II) is produced as asecretable factor (Tone, M., et al., 2001). Type II CD40 may act as adominant negative receptor, downregulating signaling through CD40L andpotentially limiting the potency of the immune response generated.Therefore, the present invention co-opts the natural regulation of CD40by creating an inducible form of CD40 (iCD40), lacking the extracellulardomain and activated instead by synthetic dimerizing ligands (Spencer,D. M. et al., 1993) through a technology termed chemically induceddimerization (CID).

The present invention comprises a method of enhancing the immuneresponse in an subject comprising the step of administering either theexpression vector, expression construct or transduced antigen-presentingcells of the present invention to the subject. The expression vector ofthe present invention encodes a co-stimulatory polypeptide, such asiCD40.

In certain embodiments the antigen-presenting cells are comprised in ananimal, such as human, non-human primate, cow, horse, pig, sheep, goat,dog, cat, or rodent. The subject is a human, more preferably, a patientsuffering from an infectious disease, and/or a subject that isimmunocompromised, or is suffering from a hyperproliferative disease.

In further embodiments of the present invention, the expressionconstruct and/or expression vector can be utilized as a composition orsubstance that activates antigen-presenting cells. Such a compositionthat “activates antigen-presenting cells” or “enhances the activityantigen-presenting cells” refers to the ability to stimulate one or moreactivities associated with antigen-presenting cells. Such activities arewell known by those of skill in the art. For example, a composition,such as the expression construct or vector of the present invention, canstimulate upregulation of co-stimulatory molecules on antigen presentingcells, induce nuclear translocation of NF-κB in antigen presentingcells, activate toll-like receptors in antigen presenting cells, orother activities involving cytokines or chemokines.

An amount of a composition that activates antigen-presenting cells which“enhances” an immune response refers to an amount in which an immuneresponse is observed that is greater or intensified or deviated in anyway with the addition of the composition when compared to the sameimmune response measured without the addition of the composition. Forexample, the lytic activity of cytotoxic T cells can be measured, e.g.,using a ⁵¹Cr release assay, with and without the composition. The amountof the substance at which the CTL lytic activity is enhanced as comparedto the CTL lytic activity without the composition is said to be anamount sufficient to enhance the immune response of the animal to theantigen. In a preferred embodiment, the immune response in enhanced by afactor of at least about 2, more preferably by a factor of about 3 ormore. The amount of cytokines secreted may also be altered.

The enhanced immune response may be an active or a passive immuneresponse. Alternatively, the response may be part of an adaptiveimmunotherapy approach in which antigen-presenting cells are obtainedwith from a subject (e.g., a patient), then transduced with acomposition comprising the expression vector or construct of the presentinvention. The antigen-presenting cells may be obtained from the bloodof the subject or bone marrow of the subject. In certain preferredembodiments, the antigen-presenting cells are isolated from the bonemarrow. In a preferred embodiment, the antigen-presenting cells areadministered to the same or different animal (e.g., same or differentdonors). In a preferred embodiment, the subject (e.g., a patient) has oris suspected of having a cancer, such as for example, prostate cancer,or has or is suspected of having an infectious disease. In otherembodiments the method of enhancing the immune response is practiced inconjunction with a known cancer therapy or any known therapy to treatthe infectious disease.

The expression construct, expression vector and/or transducedantigen-presenting cells can enhance or contribute to the effectivenessof a vaccine by, for example, enhancing the immunogenicity of weakerantigens such as highly purified or recombinant antigens, reducing theamount of antigen required for an immune response, reducing thefrequency of immunization required to provide protective immunity,improve the efficacy of vaccines in subjects with reduced or weakenedimmune responses, such as newborns, the aged, and immunocompromisedindividuals, and enhance the immunity at a target tissue, such asmucosal immunity, or promote cell-mediated or humoral immunity byeliciting a particular cytokine profile.

Yet further, an immunocompromised individual or subject is a subjectthat has a reduced or weakened immune response. Such individuals mayalso include a subject that has undergone chemotherapy or any othertherapy resulting in a weakened immune system, a transplant recipient, asubject currently taking immunosuppressants, an aging individual, or anyindividual that has a reduced and/or impaired CD4 T helper cells. It iscontemplated that the present invention can be utilize to ehance theamount and/or activty of CD4 T helper cells in an immunocompromisedsubject.

In specific embodiments, prior to administering the transducedantigen-presenting cell, the cells are challenged with antigens (alsoreferred herein as “target antigens”). After challenge, the transduced,loaded antigen-presenting cells are administered to the subjectparenterally, intradermally, intranodally, or intralymphatically.Additional parenteral routes include, but are not limited tosubcutaneous, intramuscular, intraperitoneal, intravenous,intraarterial, intramyocardial, transendocardial, transepicardial,intrathecal, and infusion techniques.

The target antigen, as used herein, is an antigen or immunologicalepitope on the antigen, which is crucial in immune recognition andultimate elimination or control of the disease-causing agent or diseasestate in a mammal The immune recognition may be cellular and/or humoral.In the case of intracellular pathogens and cancer, immune recognition ispreferably a T lymphocyte response.

The target antigen may be derived or isolated from a pathogenicmicroorganism such as viruses including HIV, (Korber et al, 1977)influenza, Herpes simplex, human papilloma virus (U.S. Pat. No.5,719,054), Hepatitis B (U.S. Pat. No. 5,780,036), Hepatitis C (U.S.Pat. No. 5,709,995), EBV, Cytomegalovirus (CMV) and the like. Targetantigen may be derived or isolated from pathogenic bacteria such as fromChlamydia (U.S. Pat. No. 5,869,608), Mycobacteria, Legionella,Meningiococcus, Group A Streptococcus, Salmonella, Listeria, Hemophilusinfluenzae (U.S. Pat. No. 5,955,596) and the like.

Target antigen may be derived or isolated from pathogenic yeastincluding Aspergillus, invasive Candida (U.S. Pat. No. 5,645,992),Nocardia, Histoplasmosis, Cryptosporidia and the like.

Target antigen may be derived or isolated from a pathogenic protozoanand pathogenic parasites including but not limited to Pneumocystiscarinii, Trypanosoma, Leishmania (U.S. Pat. No. 5,965,242), Plasmodium(U.S. Pat. No. 5,589,343) and Toxoplasma gondii.

Target antigen includes an antigen associated with a preneoplastic orhyperplastic state. Target antigen may also be associated with, orcausative of cancer. Such target antigen may be tumor specific antigen,tumor associated antigen (TAA) or tissue specific antigen, epitopethereof, and epitope agonist thereof. Such target antigens include butare not limited to carcinoembryonic antigen (CEA) and epitopes thereofsuch as CAP-1, CAP-1-6D (46) and the like (GenBank Accession No.M29540), MART-1 (Kawakami et al, 1994), MAGE-1 (U.S. Pat. No.5,750,395), MAGE-3, GAGE (U.S. Pat. No. 5,648,226), GP-100 (Kawakami etal., 1992), MUC-1, MUC-2, point mutated ras oncogene, normal and pointmutated p53 oncogenes (Hollstein et al., 1994), PSMA (Israeli et al.,1993), tyrosinase (Kwon et al. 1987) TRP-1 (gp75) (Cohen et al., 1990;U.S. Pat. No. 5,840,839), NY-ESO-1 (Chen et al., PNAS 1997), TRP-2(Jackson et al., 1992), TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2,(U.S. Pat. No. 5,550,214), BRC—I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1,modifications of TAAs and tissue specific antigen, splice variants ofTAAs, epitope agonists, and the like. Other TAAs may be identified,isolated and cloned by methods known in the art such as those disclosedin U.S. Pat. No. 4,514,506. Target antigen may also include one or moregrowth factors and splice variants of each.

For organisms that contain a DNA genome, a gene encoding a targetantigen or immunological epitope thereof of interest is isolated fromthe genomic DNA. For organisms with RNA genomes, the desired gene may beisolated from cDNA copies of the genome. If restriction maps of thegenome are available, the DNA fragment that contains the gene ofinterest is cleaved by restriction endonuclease digestion by methodsroutine in the art. In instances where the desired gene has beenpreviously cloned, the genes may be readily obtained from the availableclones. Alternatively, if the DNA sequence of the gene is known, thegene can be synthesized by any of the conventional techniques forsynthesis of deoxyribonucleic acids.

Genes encoding an antigen of interest can be amplified by cloning thegene into a bacterial host. For this purpose, various prokaryoticcloning vectors can be used. Examples are plasmids pBR322, pUC andpEMBL.

The genes encoding at least one target antigen or immunological epitopethereof can be prepared for insertion into the plasmid vectors designedfor recombination with a virus by standard techniques. In general, thecloned genes can be excised from the prokaryotic cloning vector byrestriction enzyme digestion. In most cases, the excised fragment willcontain the entire coding region of the gene. The DNA fragment carryingthe cloned gene can be modified as needed, for example, to make the endsof the fragment compatible with the insertion sites of the DNA vectorsused for recombination with a virus, then purified prior to insertioninto the vectors at restriction endonuclease cleavage sites (cloningsites).

Antigen loading of dendritic cells with antigens may be achieved byincubating dendritic cells or progenitor cells with the polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide. Antigens from cells or MHC molecules may be obtained byacid-elution or other methods known in the art (see Zitvogel et al.,1996).

One skilled in the art is fully aware that activation of theco-stimulatory molecule of the present invention relies uponoligomerization of ligand-binding domains, for example CID, to induceits activity. In specific embodiments, the ligand is a non-protein. Morespecifically, the ligand is a dimeric FK506 or dimeric FK506 analogs,which result in enhancement or positive regulation of the immuneresponse. The use of monomeric FK506 or monomeric FK506 analogs resultsin inhibition or reduction in the immune response negatively.

T-lymphocytes are activated by contact with the antigen-presenting cellthat comprises the expression vector of the present invention and hasbeen challenged, transfected, pulsed, or electrofused with an antigen.

Electrofusing in the present invention is a method of generating hybridcells. There are several advantages in producing cell hybrids byelectrofusion. For example, fusion parameters can be easily andaccurately electronically controlled to conditions depending on thecells to be fused. Further, electrofusion of cells has shown to theability to increase fusion efficiency over that of fusion by chemicalmeans or via biological fusogens. Electrofusion is performed by applyingelectric pulses to cells in suspension. By exposing cells to analternating electric field, cells are brought close to each other informing pearl chains in a process termed dielectrophoresis alignment.Subsequent higher voltage pulses cause cells to come into closercontact, reversible electropores are formed in reversibly permeabilizingand mechanically breaking down cell membranes, resulting in fusion.

T cells express a unique antigen binding receptor on their membrane(T-cell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines, that are crucial for the activation of B cells, T cytotoxiccells, macrophages and other cells of the immune system. In contrast, anaïve CD8 T cell that recognizes an antigen-MHC complex proliferates anddifferentiates into an effector cell called a cytotoxic CD8 T lymphocyte(CTL). CTLs eliminate cells of the body displaying antigen, such asvirus-infected cells and tumor cells, by producing substances thatresult in cell lysis.

CTL activity can be assessed by methods described herein or as would beknown to one of skill in the art. For example, CTLs may be assessed infreshly isolated peripheral blood mononuclear cells (PBMC), in aphytohaemaglutinin-stimulated IL-2 expanded cell line established fromPBMC (Bernard et al., 1998) or by T cells isolated from a previouslyimmunized subject and restimulated for 6 days with DC infected with anadenovirus vector containing antigen using standard 4 h ⁵¹Cr releasemicrotoxicity assays. One type of assay uses cloned T-cells. ClonedT-cells have been tested for their ability to mediate both perforin andFas ligand-dependent killing in redirected cytotoxicity assays (Simpsonet al., 1998). The cloned cytotoxic T lymphocytes displayed both Fas-and perforin-dependent killing. Recently, an in vitro dehydrogenaserelease assay has been developed that takes advantage of a newfluorescent amplification system (Page et al., 1998). This approach issensitive, rapid, and reproducible and may be used advantageously formixed lymphocyte reaction (MLR). It may easily be further automated forlarge-scale cytotoxicity testing using cell membrane integrity, and isthus considered in the present invention. In another fluorometric assaydeveloped for detecting cell-mediated cytotoxicity, the fluorophore usedis the non-toxic molecule AlamarBlue (Nociari et al., 1998). TheAlamarBlue is fluorescently quenched (i.e., low quantum yield) untilmitochondrial reduction occurs, which then results in a dramaticincrease in the AlamarBlue fluorescence intensity (i.e., increase in thequantum yield). This assay is reported to be extremely sensitive,specific and requires a significantly lower number of effector cellsthan the standard ⁵¹Cr release assay.

Other immune cells that are induced by the present invention includenatural killer cells (NK). NKs are lymphoid cells that lackantigen-specific receptors and are part of the innate immune system.Typically, infected cells are usually destroyed by T cells alerted byforeign particles bound the cell surface MHC. However, virus-infectedcells signal infection by expressing viral proteins that are recognizedby antibodies. These cells can be killed by NKs. In tumor cells, if thetumor cells lose expression of MHC I molecules, then it may besusceptible to NKs.

In further embodiments, the transduced antigen-presenting cell istransfected with tumor cell mRNA. The transduced transfectedantigen-presenting cell is administered to an animal to effect cytotoxicT lymphocytes and natural killer cell anti-tumor antigen immune responseand regulated using dimeric FK506 and dimeric FK506 analogs. The tumorcell mRNA is mRNA from a prostate tumor cell.

Yet further, the transduced antigen-presenting cell is pulsed with tumorcell lysates. The pulsed transduced antigen-presenting cells areadministered to an animal to effect cytotoxic T lymphocytes and naturalkiller cell anti-tumor antigen immune response and regulated usingdimeric FK506 and dimeric FK506 analogs. The tumor cell lysates is aprostate tumor cell lysate.

VI. FORMULATIONS AND ROUTES FOR ADMINISTRATION TO PATIENTS

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression constructs, expressionvectors, fused proteins, transduced cells, activated DCs, transduced andloaded DCs—in a form appropriate for the intended application.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. A pharmaceuticallyacceptable carrier includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well know in the art. Exceptinsofar as any conventional media or agent is incompatible with thevectors or cells of the present invention, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration, the compositions of the present invention maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientalso may be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by FDAOffice of Biologics standards.

VII. METHODS FOR TREATING A DISEASE

The present invention also encompasses methods of treatment orprevention of a disease caused by pathogenic microorganisms and/or ahyperproliferative disease.

Diseases may be treated or prevented by use of the present inventioninclude diseases caused by viruses, bacteria, yeast, parasites,protozoa, cancer cells and the like. The pharmaceutical composition ofthe present invention (transduced DCs, expression vector, expressionconstruct, etc.) of the present invention may be used as a generalizedimmune enhancer (DC activating composition or system) and as such hasutility in treating diseases. Exemplary disease that can be treatedand/or prevented utilizing the pharmaceutical composition of the presentinvention include, but are not limited to infections of viral etiologysuch as HIV, influenza, Herpes, viral hepatitis, Epstein Bar, polio,viral encephalitis, measles, chicken pox, Papilloma virus etc.; orinfections of bacterial etiology such as pneumonia, tuberculosis,syphilis, etc.; or infections of parasitic etiology such as malaria,trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc.

Preneoplastic or hyperplastic states which may be treated or preventedusing the pharmaceutical composition of the present invention(transduced DCs, expression vector, expression construct, etc.) of thepresent invention include but are not limited to preneoplastic orhyperplastic states such as colon polyps, Crohn's disease, ulcerativecolitis, breast lesions and the like.

Cancers which may be treated using the pharmaceutical composition of thepresent invention of the present invention include, but are not limitedto primary or metastatic melanoma, adenocarcinoma, squamous cellcarcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma,lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma,leukemias, uterine cancer, breast cancer, prostate cancer, ovariancancer, pancreatic cancer, colon cancer, multiple myeloma,neuroblastoma, NPC, bladder cancer, cervical cancer and the like.

Other hyperproliferative diseases that may be treated using DCactivation system of the present invention include, but are not limitedto rheumatoid arthritis, inflammatory bowel disease, osteoarthritis,leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascularocclusion, restenosis, atherosclerosis, pre-neoplastic lesions (such asadenomatous hyperplasia and prostatic intraepithelial neoplasia),carcinoma in situ, oral hairy leukoplakia, or psoriasis.

In the method of treatment, the administration of the pharmaceuticalcomposition (expression construct, expression vector, fused protein,transduced cells, activated DCs, transduced and loaded DCs) of theinvention may be for either “prophylactic” or “therapeutic” purpose.When provided prophylactically, the pharmaceutical composition of thepresent invention is provided in advance of any symptom. Theprophylactic administration of pharmaceutical composition serves toprevent or ameliorate any subsequent infection or disease. When providedtherapeutically, the pharmaceutical composition is provided at or afterthe onset of a symptom of infection or disease. Thus the presentinvention may be provided either prior to the anticipated exposure to adisease-causing agent or disease state or after the initiation of theinfection or disease.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of pharmaceutical compositioncalculated to produce the desired immunogenic effect in association withthe required diluent. The specifications for the novel unit dose of aninoculum of this invention are dictated by and are dependent upon theunique characteristics of the pharmaceutical composition and theparticular immunologic effect to be achieved.

An effective amount of the pharmaceutical composition would be theamount that achieves this selected result of enhancing the immuneresponse, and such an amount could be determined as a matter of routineby a person skilled in the art. For example, an effective amount of fortreating an immune system deficiency could be that amount necessary tocause activation of the immune system, resulting in the development ofan antigen specific immune response upon exposure to antigen. The termis also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

A. Genetic Based Therapies

Specifically, the present inventors intend to provide, to a cell, anexpression construct capable of providing a co-stimulatory polypeptide,such as CD40 to the cell, such as an antigen-presenting cell andactivating CD40. The lengthy discussion of expression vectors and thegenetic elements employed therein is incorporated into this section byreference. Particularly preferred expression vectors are viral vectorssuch as adenovirus, adeno-associated virus, herpes virus, vaccinia virusand retrovirus. Also preferred is lysosomal-encapsulated expressionvector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

B. Cell Based Therapy

Another therapy that is contemplated is the administration of transducedantigen-presenting cells. The antigen-presenting cells may be transducedin vitro. Formulation as a pharmaceutically acceptable composition isdiscussed above.

In cell based therapies, the transduced antigen-presenting cells may betransfected with target antigen nucleic acids, such as mRNA or DNA orproteins; pulsed with cell lysates, proteins or nucleic acids; orelectrofused with cells. The cells, proteins, cell lysates, or nucleicacid may derive from cells, such as tumor cells or other pathogenicmicroorganism, for example, viruses, bacteria, protozoa, etc.

C. Combination Therapies

In order to increase the effectiveness of the expression vector of thepresent invention, it may be desirable to combine these compositions andmethods of the invention with an agent effective in the treatment of thedisease.

In certain embodiments, anti-cancer agents may be used in combinationwith the present invention. An “anti-cancer” agent is capable ofnegatively affecting cancer in a subject, for example, by killing one ormore cancer cells, inducing apoptosis in one or more cancer cells,reducing the growth rate of one or more cancer cells, reducing theincidence or number of metastases, reducing a tumor's size, inhibiting atumor's growth, reducing the blood supply to a tumor or one or morecancer cells, promoting an immune response against one or more cancercells or a tumor, preventing or inhibiting the progression of a cancer,or increasing the lifespan of a subject with a cancer. Anti-canceragents include, for example, chemotherapy agents (chemotherapy),radiotherapy agents (radiotherapy), a surgical procedure (surgery),immune therapy agents (immunotherapy), genetic therapy agents (genetherapy), hormonal therapy, other biological agents (biotherapy) and/oralternative therapies.

In further embodiments antibiotics can be used in combination with thepharmaceutical composition of the present invention to treat and/orprevent an infectious disease. Such antibiotics include, but are notlimited to, amikacin, aminoglycosides (e.g., gentamycin), amoxicillin,amphotericin B, ampicillin, antimonials, atovaquone sodiumstibogluconate, azithromycin, capreomycin, cefotaxime, cefoxitin,ceftriaxone, chloramphenicol, clarithromycin, clindamycin, clofazimine,cycloserine, dapsone, doxycycline, ethambutol, ethionamide, fluconazole,fluoroquinolones, isoniazid, itraconazole, kanamycin, ketoconazole,minocycline, ofloxacin), para-aminosalicylic acid, pentamidine,polymixin definsins, prothionamide, pyrazinamide, pyrimethaminesulfadiazine, quinolones (e.g., ciprofloxacin), rifabutin, rifampin,sparfloxacin, streptomycin, sulfonamides, tetracyclines, thiacetazone,trimethaprim-sulfamethoxazole, viomycin or combinations thereof.

More generally, such an agent would be provided in a combined amountwith the expression vector effective to kill or inhibit proliferation ofa cancer cell and/or microorganism. This process may involve contactingthe cell(s) with an agent(s) and the pharmaceutical composition of thepresent invention at the same time or within a period of time whereinseparate administration of the pharmaceutical composition of the presentinvention and an agent to a cell, tissue or organism produces a desiredtherapeutic benefit. This may be achieved by contacting the cell, tissueor organism with a single composition or pharmacological formulationthat includes both the pharmaceutical composition of the presentinvention and one or more agents, or by contacting the cell with two ormore distinct compositions or formulations, wherein one compositionincludes the pharmaceutical composition of the present invention and theother includes one or more agents.

The terms “contacted” and “exposed,” when applied to a cell, tissue ororganism, are used herein to describe the process by which thepharmaceutical composition and/or another agent, such as for example achemotherapeutic or radiotherapeutic agent, are delivered to a targetcell, tissue or organism or are placed in direct juxtaposition with thetarget cell, tissue or organism. To achieve cell killing or stasis, thepharmaceutical composition and/or additional agent(s) are delivered toone or more cells in a combined amount effective to kill the cell(s) orprevent them from dividing.

The administration of the pharmaceutical composition may precede, beco-current with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the pharmaceuticalcomposition of the present invention, and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the times ofeach delivery, such that the pharmaceutical composition of the presentinvention and agent(s) would still be able to exert an advantageouslycombined effect on the cell, tissue or organism. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) as thepharmaceutical composition of the present invention. In other aspects,one or more agents may be administered within of from substantiallysimultaneously, about 1 minute, to about 24 hours to about 7 days toabout 1 to about 8 weeks or more, and any range derivable therein, priorto and/or after administering the expression vector. Yet further,various combination regimens of the pharmaceutical composition of thepresent invention and one or more agents may be employed.

VIII. TRANSGENIC ANIMALS

Detailed methods for generating non-human transgenic animal aredescribed herein. Any non-human animal can be used in the methodsdescribed herein. Preferred mammals are rodents, e.g., rats or mice.

A transgenic mouse describes an mouse that has had genes from anotherorganism inserted into its genome through recombinant DNA techniques.The transgenic mouse may contain material from an unrelated organism,such as from a virus, plant, or human. Thus, in an exemplary embodiment,the “transgenic non-human animals” of the invention are produced byintroducing transgenes into the germline of the non-human animal.

Introduction of the transgene into the embryo can be accomplished by anymeans known in the art such as, for example, microinjection,electroporation, or lipofection. For example, the CD40 gene can beintroduced into a mammal by microinjection of the construct into thepronuclei of the fertilized mammalian egg(s) to cause one or more copiesof the construct to be retained in the cells of the developingmammal(s). Following introduction of the transgene construct into thefertilized egg, the egg may be incubated in vitro for varying amounts oftime, or reimplanted into the surrogate host, or both. In vitroincubation to maturity is within the scope of this invention. One commonmethod is to incubate the embryos in vitro for about 1-7 days, dependingon the species, and then reimplant them into the surrogate host.

Embryonic target cells at various developmental stages can also be usedto introduce transgenes. Different methods are used depending on thestage of development of the embryonic target cell. The specific line(s)of any animal used to practice this invention are selected for generalgood health, good embryo yields, good pronuclear visibility in theembryo, and good reproductive fitness. In addition, the haplotype is asignificant factor.

Embryonic stem cells, sometimes referred to as ES cells, are derivedfrom inner cell mass (ICM) of fertilized eggs in blastocyst phase, andcan be cultured and maintained in vitro while being kept in anundifferentiated state. Embryonic stem cells are extremely usefulbiological materials for preparing transgenic animals. For example, agene knockout mouse in which a specific gene is inactivated can beproduced by replacing an active gene in an embryonic stem cellchromosome with an inactivated gene by means of a homologousrecombination system.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the CD40 construct by Southern blot analysis of thesegment of tissue. If one or more copies of the exogenous clonedconstruct remains stably integrated into the genome of such transgenicembryos, it is possible to establish permanent transgenic mammal linescarrying the transgenically added construct.

The litters of transgenically altered mammals can be assayed after birthfor the incorporation of the construct into the genome of the offspring.Preferably, this assay is accomplished by hybridizing a probecorresponding to the DNA sequence coding for the desired recombinantprotein product or a segment thereof onto chromosomal material from theprogeny. Those mammalian progeny found to contain at least one copy ofthe construct in their genome are grown to maturity.

In certain embodiments of the invention, transgenic mice are producedwhich contain an expression vector comprising a polynucleotide promotersequence, a polynucleotide sequence encoding a CD40 cytoplasmic domainand a polynucleotide sequence encoding a dimeric ligand-binding region,all operatively linked. These mice may be used to obtainantigen-presenting cells, such as dendritic cells, which express theCD40 cytoplasmic domain.

IX. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Development of Techniques for Efficient Isolation of DCs

Bone marrow from the tibias and femurs of C57BL/6 mice was cultured inRPMI supplemented with GM-CSF and IL-4 (Inaba, K. et al., 1992). Bonemarrow cultures were maintained for a total of 6 days in 24 well plateswhile one-half of each well was replenished with fresh media andcytokines on day 3. On the final day of culture, cells were washed fromthe plates, co-incubated with anti-CD11c microbeads (Miltenyi Biotec,Bergisch Gladbach, Germany), and applied to two consecutive magneticcolumns Splenic DCs were subjected to microbead purification immediatelyfollowing collagenase treatment of splenic tissue. Based on flowcytometry analysis, the DC purity of the final cell suspension wasconsistently >95% for bone marrow-derived DCs and >80% for splenic DCs.

Example 2 Development of Techniques for Efficient Cellular Electrofusion

Two Jurkat T cell populations were individually stained with distinctlipophilic dyes, fluorescing at different wavelengths (DiI and DiO).These cell populations were mixed and fused using the BTX2001electrofusion instrument at different D.C. voltages. Fusion efficiencywas analyzed by flow cytometry. Electrofusion of Jurkat TAg cells usinga 275 V D.C. generated viable hybrid yields of around 60%.

Example 3 Construction of an Inducible CD40 Vector

An inducible CD40 receptor based on chemical-induced dimerization (CID)and patterned after endogenous CD40 activation was produced tospecifically target DCs (FIG. 1A). The recombinant CD40 receptor, termediCD40, was engineered by rt-PCR amplifying the 228 by CD40 cytoplasmicsignaling domain from purified murine bone marrow-derived DCs (>95%CD11c+) and sub-cloning the resulting DNA fragment either downstream(i.e., M-FvFv1sCD40-E) or upstream (M-CD40-FvFvls-E) of tandem copies ofthe dimerizing drug binding domain, FKBP12(V36) (FIG. 1B). Membranelocalization was achieved with a myristoylation-targeting domain (M) andan HA epitope (E) tag was present for facile identification. Todetermine if the transcripts were capable of activating NFκB, theconstructs were transiently transfected into Jurkat T cells and NFκBreporter assays were preformed in the presence of titrated dimerizerdrug, AP20187 (FIG. 1C). FIG. 1C showed that increasing levels ofAP20187 resulted in significant upregulation of NFκB transcriptionalactivity compared to the control vector, M-FvFvls-E, lacking CD40sequence. Since the membrane-proximal version of iCD40, M-CD40-FvFvls-E,was less responsive to AP20187 in this assay system, the M-FvFvlsCD40-Econstruct was used in further studies, and heretofore referred to as“iCD40”. This decision was reinforced by the crystallographic structureof the CD40 cytoplasmic tail, which reveals a hairpin conformation thatcould be deleteriously altered by the fusion of a heterologous proteinto its carboxyl-terminus (Ni 2000). The data also showed high drug dosesuppression over 100 nM, likely due to the saturation of drug bindingdomains. This same phenomenon has been observed in other cell typesexpressing limiting levels of the iCD40 receptor. These resultssuggested that iCD40 was capable of inducing CID-dependent nucleartranslocation of the NF1B transcription factor.

Once the Ad-iCD40-GFP virus was demonstrated to successfully transduce293T cells and to induce the expression of both iCD40 and GFPtransgenes, bone marrow-derived dendritic cells were transduced withefficiencies ranging between 25-50% under serum-free conditions. Theresults have demonstrated that iCD40-expressing primary DCs exhibited anupregulation in maturation markers (e.g., CD86, CD40) and an enhancedability to synthesize the IL-12 cytokine when treated with CID. Inaddition, CID-treated iCD40-expressing primary DCs survived longer inculture and were capable of inducing a more robust CTL response in vivocompared to non-transduced DC and Ad-GFP-transduced DC controls.

Example 4 Cell Fusion

TRAMP-C2 murine prostate cancer cell line is cultured in high glucosemedia with insulin and DHT and with Cell Tracker Green Dye (CMFDA,Molecular Probes, Eugene, Oreg.). Isolated DCs that are stained withCell Tracker Orange Dye (CMTMR, Molecular Probes, Eugene, Oreg.) arefused to g-irradiated TRAMP-C2 cells using the lab's BTX Electro-CellManipulator 2001 instrument (Genetronics, San Diego, Calif.), whichapplies an alternating current to dimerize cells at the center of anelectrical field. A high voltage direct current pulse is then releasedwhich fuses the membranes of the dimerized cells, subjecting cells toharsh fusogenic conditions for shorter periods of time and producinghigher hybrid yields than the standard polyethylene glycol fusion.Although irradiated, the hybrid cells survive for several days.

Example 5 CTL/IFN-γ Assay

Splenocytes and 4-6 lymph nodes are incubated with mitomycin C-treated(and washed 8×) TRAMP-B7 cells (obtained from Jim Allison, UCB). After 7days expanded/viable T cells are Ficoll-purified and stimulated withTRAMP-C2-B7 a second time. After 7 additional days, dilutions ofTRAMP-C2 cells (pre-incubated for 2 days with 1 ng/ml IFNγ to boost MHC)are incubated with T cells and analyzed for de novo IFNγ production.Alternatively, target cells will be pre-loaded with ⁵¹Cr for true CTLassays.

Example 6 Determine Whether iCD40 Activation In Situ can IncreaseAnti-Tumor Immunity

Cultured proliferating DCs are transduced with adenovirus expressingiCD40 and then fused with irradiated TRAMP-C2 cells, as before. For −6 htimepoints, DC:TRAMP heterokaryon are maintained in culture mediasupplemented with the dimerizer AP20187 for 6 hours prior to vaccinepreparation and administration. For all other CID timepoints (0, 6, 12and 24 h post-vaccination), dimerizer AP20187 are delivered tovaccinated and control mice by i.p. injection. An additional controlgroup receives DC/iCD40:TRAMP hybrids but no CID. Two weeks after abooster vaccine, mice will be challenged with 2×10⁶ s.c. TRAMP-C2 cellsas before. The efficacy of iCD40 stimulation in situ versus in vitro iscorrelated with measurements of tumor incidence and size and by CTLassays after DC/iCD40:tumor vaccination in the presence or absence ofCID.

Example 7 Transfection of DCs with mRNAs from Tumor Cells

Using the methods of Gilboa and colleagues (Gilboa, et al., 1998;Boczkowski, D., et al., 1996), cDNAs made from TRAMP-C2 cells areamplified and subcloned into an expression vector. Followingtranscription in a reticulocyte lysate, mRNA is purified using a poly-Tprimer and magnetic bead separation. A number of lipid-basedtransfection protocols (e.g., FuGene6, Superfectin) are currentlyavailable, and the most effective method based on control transfectionsof freshly amplified DCs using a reporter plasmid are used.

Example 8 Pulsing of DCs with Peptides from Tumor Cells

To increase the likelihood of transferring relevant tumor-derivedpeptides, capable of binding to MHC molecules, DCs are pulsed withpeptides derived from TRAMP-C2 cells. Since MHC levels are extremely lowon cultured TRAMP-derived cells, MHC levels on tumor cells are boostedusing 5 ng/ml murine IFN-γ. MHC-derived peptides are purified using HPLCand acid-treated MHC using previously described methods (Nair, S. K. etal., 1997). Finally, DCs are initially treated with anti-sense againstthe TAP peptide transporter to increase the density of “empty” MHC onthe surface as previously described (Nair, S. K. et al., 1996).

Example 9 Pulsing of DCs with Other Antigens

Active specific immunotherapy using vaccines consisting of isolatedmurine tumor-derived HSPPC-96 was demonstrated against murine tumors(Tamura, Y. et al., 1997). The gp96 itself is non-polymorphic but actsas a chaperone for tightly bound immunogenic peptides, which arebelieved to represent the full cellular repertoire of immunogens. Thus,gp96 proteins are purified using an affinity column and used to primeDCs.

Example 10 iCD40 Activates NFκB in DCs

The physiological response of iCD40 was evaluated in the immature DCline, D2SC/1 (Lutz 1994), which maintains a stable phenotype overprolonged culture periods. The use of this DC line avoided confoundingmaturation effects that often occur spontaneously in primary DCs duringtypical culturing conditions. iCD40 was subcloned into a bicistronicvector co-expressing the Neo^(R) gene and the resulting vector waselectroporated into D2SC/1 cells. D2SC/1 DCs stably expressing the iCD40transgene were selected in culture by G418, and clonal lines werederived by limiting dilution. Screening by probing for the HA-epitopetag allowed for high (Hi), intermediate (Int), and low (Lo)iCD40-expressing D2SC/1 DC clones (iCD40 DCs) to be selected for furtheranalysis (FIG. 2A). Additional immunofluorescence experiments wereperformed to verify the membrane localization of iCD40 (FIG. 2B).

Reporter assays were carried out in these cell lines to determinewhether iCD40 could also induce NFκB activation in DCs. IncreasingAP20187 concentrations resulted in consistent elevation of NFκB activitythat was further reflected in the levels of transgene expression by therespective DC lines (FIG. 2C). Several studies have identified that theRelB subunit of NFκB plays a significant role in the DC maturationprogram (Pettit 1997; Martin 2003). Indeed, nuclear localization of RelBcorrelated with the mature DC state and Rel^(−/−) DCs exhibited aconstitutively immature phenotype. Therefore, as a surrogate marker forDC activation, the nuclear translocation of RelB was analyzed by westernblot in iCD40 DCs exposed to log dilutions of AP20187 (FIG. 2D).Additional results further confirmed that iCD40 triggered the nucleartranslocation of DC-expressed RelB in a CID-dependent manner.

DC activation potency of iCD40 was compared with other traditional DCmaturation stimuli, such as LPS, TNFα, anti-CD40 mAb, and CD40L. For themost informative comparison, each agent was titrated to determine theoptimal concentration for RelB induction in iCD40 DCs and theseconcentrations were utilized to directly compare RelB activation bythese different factors (FIG. 2E). The data clearly indicated thatdrug-dependent RelB induction in iCD40 DCs is superior to LPS, TNFα,anti-CD40 mAb, and CD40L. Pulse-chase experiments further revealed thatthe AP20187 dimerizer drug stimulated a more prolonged and durable RelBnuclear signal than TNFα or the anti-CD40 mAb (FIG. 2F). WhileTNFα-mediated RelB induction terminated after 24 hrs and anti-CD40 mAbinduced RelB up to 48 hrs, the iCD40 receptor stimulated the RelBmaturation pathway for at least 72 hrs in the presence of dimerizerdrug. These results suggested that upon drug-mediated stimulation, iCD40DCs are capable of maintaining a hyper-extended activation state.

Example 11 iCD40 Induces DC Activation

A component of the DC maturation program includes the upregulation ofseveral surface molecules that participate in the process of T cellstimulation by direct involvement in antigen presentation andcostimulation. Therefore, iCD40 DCs were treated with AP20187 and thesurface expression of several of maturation markers, including, ICAM-1(CD54), B7.1 (CD80), MHC class I K^(d), MHC class II I-A^(d) andendogenous CD40 (FIG. 3A) were analyzed by flow cytometry. Exposure ofthese DCs to CID resulted in significant elevations in the expression ofeach of these immunostimulatory proteins over untreated iCD40 DCs andthe parental D2SC/I line. This observed increase in the fluorescenceintensity of these mature DC markers was comparable to that of LPS (fromE. coli)-treated DCs. Only minimal basal signaling of the iCD40 receptorwas detectable in untreated iCD40 DCs and drug treatment of the parentalcontrol D2SC/I line had no observable effect. Moreover, when iCD40 DCswere pre-treated with an excess of a monomeric form of the drug,dimerizer drug-dependent upregulation of these surface markers wascompletely abolished, indicating that physical aggregation of the CD40cytoplasmic domain was absolutely required for inducing the mature DCphenotype in the D2SC/I line.

When DCs undergo maturation, they also exhibit functional alterations.These changes include a reduced capacity to uptake molecules from theirmicroenvironment and a concomitant enhancement of their ability tostimulate T cell activation. Drug-induced modification of D2SC/1DCreceptor-mediated endocytosis was investigated by measuring the uptakeof a FITC-tagged dextran molecule in iCD40 DCs and the parental D2SC/Iline. (FIG. 3B). CID-mediated activation of iCD40 DCs resulted in thereduced uptake of FITC-dextran to levels comparable to that ofLPS-treated DCs at 37° C. Performing this same series of experiments at0° C. resulted in minimal uptake of the FITC-dextran molecule,confirming that iCD40 activation is also capable of regulating theantigen uptake function of this DC line.

The initial approach to the determination of the T cell stimulationcapacity of iCD40 DCs involved the co-incubation of mitomycin C-treatedD2SC/1 DCs with syngeneic lymph node (LN)-derived cells in vitro. Thismixed lymphocyte reaction (MLR) assay measured the ability of iCD40 DCsto induce a syngeneic T cell proliferative response to bovineserum-derived xenogeneic antigens (FIG. 3C). The results indicated thatCID-treated iCD40 DCs were capable of inducing T cell proliferation tolevels similar to that of LPS-treated DCs in vitro. In order toinvestigate whether this T cell activation effect was dependent on CD4+T cell help, CD8+ T cells from LN tissue (>95%) were purified and the³H-thymidine-based proliferation assay was repeated (FIG. 3C). The datashowed that iCD40 DCs were capable of inducing CD8+ T cell proliferationin a CD4+-independent manner as opposed to LPS-treated DCs that failedto circumvent the requirement for CD4+ T cell-derived helper signals.These results demonstrated that iCD40-expressing DCs that have beenpre-exposed to the dimerizer drug exhibited greater T cell stimulationcapacity in vitro. Furthermore, the CD4+-depletion data inferred thatthe iCD40 receptor may be capable of substituting for CD4+ T cell helpin DC-based vaccination strategies.

Example 12 In Vivo Drug-Mediated Activation of iCD40 DCs FollowingVaccination

Since in vitro conditions may not necessarily mirror the morephysiologically relevant conditions in vivo, the ability of iCD40 DCs toinduce an antigen-specific T cell response in vivo followingdrug-mediated activation pre- or post-vaccine delivery was investigated.1×10⁶ parental and iCD40-expressing D2SC/1 DCs were pulsed with theH-2K^(d)-restricted peptide antigen, LLO₉₁₋₉₉, derived from thelisteriolysin O protein of Listeria monocytogenes±LPS, or AP20187, andinjected intraperitoneally (i.p.) into syngeneic BALB/c mice. A subsetof mice receiving LLO₉₁₋₉₉-pulsed DCs, were injected i.p. with eitherAP20187 or anti-CD40 mAb ˜20 hours following the initial vaccination.Finally, splenocytes were harvested from LLO-primed mice 10 days laterand co-cultured with mitomycin C-treated LLO₉₁₋₉₉-pulsed and non-pulsedDCs for an additional 5 days before being evaluated for the uptake of³H-thymidine (FIG. 4A). The results indicated that in vivo activation ofiCD40 DCs by AP20187 injection significantly enhanced the resulting Tcell response relative to in vitro iCD40 activation prior to DC vaccinedelivery (FIG. 4B). Furthermore, the combined pre- and post-activationof iCD40 DCs resulted in an additive T cell proliferative effect,suggesting that the in vitro activation of DCs did not confer adverseeffects, such as attenuation of their migrational capacity. As opposedto the in vitro T cell proliferation assay discussed above, iCD40stimulation of DCs in vivo resulted in a significantly more robust Tcell response compared to LPS-treated DCs. Moreover, using anH-2K^(d)-LLO₉₁₋₉₉-specific tetramer, it was determined that asignificant fraction of the responding T cell population was specificfor the K^(d)-restricted peptide epitope originally used to load theDC-based vaccine (FIGS. 4C,D).

While tetramer analysis is a powerful aid in quantitating T cellpopulations, it does not reflect T cell effector function. In order toinvestigate the functionality of the T cells that respond to iCD40 DCantigen presentation, a P815-derived tumor cell line (P13.1), whichectopically expresses the β-galactosidase (βgal) protein as a surrogatetumor antigen, was utilized. After vaccinating BALB/c mice withβgal-loaded DCs and following the aforementioned culture protocol, a⁵¹Cr-release cytotoxic T lymphocyte (CTL) assay was performed to assessthe ability of the stimulated T cells to destroy βgal-expressing tumorcells. Consistent with the in vivo T cell proliferation data presentedabove, the delivery of AP20187 following DC administration resulted inimproved tumor cell killing relative to non-activated or pre-activatedDCs (FIG. 4D). To further study the CTL response to iCD40-expressingDCs, an LLO₉₁₋₉₉-expressing A20 lymphoma line was generated by cloningin-frame two tandem LLO₉₁₋₉₉ minigenes upstream of a HygGFP fusionprotein. This strategy allowed for selection of LLO₉₁₋₉₉-expressing A20tumor cells in culture with hygromycin and for tracking LLO₉₁₋₉₉expression by flow cytometry analysis of enhanced green fluorescentprotein (EGFP) (FIG. 4E). The LLO₉₁₋₉₉-expressing construct alsoincluded two adjacent AAY amino acid sequences after each minigene toimprove proteosomal processing efficiency of MHC class I-restrictedpeptides. Despite flow cytometry profiles indicating that fusion of theLLO₉₁₋₉₉ peptide upstream of HygGFP destabilized the functionality ofeither the HygR gene or the fluorescence intensity of EGFP, the A20-LLOtumor line still exhibited greatly enhanced sensitivity to CTL-mediatedkilling following priming by LLO₉₁₋₉₉-loaded AP20187-exposed iCD40 DCs.

Example 13 iCD40 Activates Primary Bone Marrow-Derived DCs

While D2SC/1 cells possess many characteristics of freshly isolated DCs,it was important to examine iCD40 functionality in primary bonemarrow-derived DCs (BMDCs) by utilizing an iCD40-expressing adenovirus.A viral region E1 and E3-deleted, replication-deficient type 5adenoviral vector was engineered to express both iCD40 and EGFP underthe control of the CMV early/immediate promoter/enhancer. Ad-iCD40-GFPsuccessfully transduced and expressed the iCD40 transgene, as well asthe EGFP marker, in purified BMDCs (FIGS. 5A,B). Titrating Ad-iCD40-GFPwhile measuring iCD40-induced upregulation of B7.2 (CD86), showed thatmaximum drug-mediated iCD40 activation occurred at around 100 moi andproceeded asymptotically to plateau at higher viral titers. Although theeffects were modest, AP20187 induced the surface expression of MHC classI K^(b), B7.2, as well as endogenous CD40 on iCD40-expressing BMDCs at100 moi but not on non-transduced DCs (FIG. 5C). The effects ofAd-iCD40-GFP on BMDCs were studied by using intracellular cytokinestaining to evaluate DC expression of the T_(H)1-polarizing cytokine,IL-12. The results confirmed numerous previous reports that an emptyadenoviral vector can contribute to background fluorescence readings bystimulating the production of low levels of this cytokine (FIG. 5D)(Korst 2002). These data also revealed that the iCD40 transgene couldgenerate a significant level of basal signaling at these titers even inthe absence of CID. However, AP20187 exposure of these iCD40-expressingDCs managed to reproducibly overcome these cumulative effects to furtherincrease the percentage of IL-12⁺ DCs. Interestingly, the stimulation ofIL-12p70/p40 synthesis with LPS and CD40L peaked at 8 hrs and decreasedthereafter, while the percentage of IL-12⁺ DCs continued to increaseuntil at least 24 hrs following adenoviral transduction. Previous workby Langenkamp et al. (Langenkamp 2000) has demonstrated that prolongedtreatment of DCs with LPS exhausted their capacity for cytokineproduction. These results imply that iCD40, as opposed to the LPS dangersignal, was capable of promoting and maintaining a more durable IL-12response by BMDCs.

In addition to DC activation state, DC longevity is another criticalvariable that influences the generation of T cell-dependent immunity. Infact, CTL-mediated killing of DCs is considered to be a significantmechanism for modulating immune responses while protecting the host fromautoimmune pathologies. Other work has established that CD40 stimulationof DCs prolongs their survival by a variety of mechanisms, includingupregulation of the anti-apoptotic protein bcl-X_(L) and the granzyme Binhibitor spi-6 (Medema 2001; Miga 2001). The effects of iCD40 relativeto CD40L on DC survival were compared in an in vitro serum-starvationculture assay (FIG. 5E). By analyzing the membrane compromised propidiumiodide (PI)-positive cell population by flow cytometry, it wasdetermined that iCD40 expressing-BMDCs exhibited greater longevity underthese conditions compared to non-transduced DCs treated with CD40L. Thiseffect was iCD40-dependent since Ad-GFP-transduced DCs failed to reflectimproved survival under these conditions. These results also showed thatexposure of iCD40 BMDCs to the AP20187 dimerizer drug even furtherenhanced this survival effect relative to untreated BMDCs.

Despite the unintended maturation induced by the adenoviral vector andthe enhanced basal signaling effects of iCD40 in primary BMDCs, enhancedDC activation in the presence of AP20187 was consistently detected.Overall, these data suggest that an inducible CD40 receptor designed torespond to a pharmacological agent was capable of maintaining primaryDCs in a sustained state of activation compared to the more transienteffects of CD40L stimulation. These data were consistent with earlierfindings describing only short-term DC modulation for stimuli thattarget endogenous CD40.

Example 14 iCD40 Activation Switch Functions as a Potent Adjuvant forDNA Vaccines

Previous studies have demonstrated that DCs play a critical role in theprocessing and presentation of DNA vaccines to responding T cells.Therefore, in order to examine the effects of iCD40 on primary DCfunctionality in vivo, the iCD40 activation switch was incorporated intoa gene gun-dependent DNA vaccination protocol (Singh 2002). Goldmicro-particles were coated with an OVA257-264 minigene plasmid in thepresence and absence of a bicistronic vector co-expressing iCD40 withthe hrGFP reporter. Biolistic transfection of C57BL/6 mice with theOVA257-264 minigene resulted in a ˜3-fold enhancement in the percent ofOVA257-264-specific CD8+ T cells, while the inclusion of theiCD40-expressing vector dramatically increased this same CD8+ T cellpopulation by an additional ˜2-fold (FIGS. 6A,C). Although AP20187 didnot further stimulate the expansion of OVA257-264-specific CD8+ T cells,i.p. administration of the dimerizer drug ˜20 hours post-vaccination didenhance the percentage of activated CD69+CD8+ T cells (FIG. 6B).

In order to demonstrate the effectiveness of the iCD40 system in theabsence of CD4+ T helper cells. Wildtype (C57BL/6) and CD4-knockout (Thelper cell deficient) mice were vaccinated using the above Gene Gun DNAvaccination protocol. FIG. 7A shows that the enhancement ofantigen-specific CD8+ T cell population existed in mice ˜14 daysfollowing vaccination. FIG. 7B shows the enhancement of activated(CD69+) CD8+ T cells ˜14 days following vaccination. All the data wasnormalized to the antigen OVA alone. The data further demonstrated theenhanced potency of the drug-regulated iCD40 system relative to thefull-length CD40 receptor and the agonistic anti-CD40 monoclonalantibody.

Thus, these results demonstrated the efficacy of the iCD40 activationswitch in vivo, and showed that iCD40 can still upregulate CD8+ T cellresponses in the absence of CD4+ T helper cells.

Example 15 Insulated iCD40 Receptor is Resistant to Ligand-InducedDownregulation and to Negative Feedback Inhibition Mediated by the TypeII CD40 Isoform

The data presented above implies that the drug-regulated iCD40 receptoris capable of delivering a more potent stimulatory signal to DCs thanthe activation of endogenous CD40. It was determined that the underlyingcause for this difference was based on the lack of an extracellulardomain, making iCD40 resistant to both ligand-induced receptordownregulation and to interference by dominant negative receptors.

To initially investigate the potential downregulation of surface CD40upon ligand engagement, flow cytometric analysis of DC surfaceexpression of CD40 was monitored in the presence and absence of CD40L.Addition of CD40L promptly reduced the mean fluorescence intensity ofCD40 on the D2SC/1 DC line with rapid kinetics Inhibition studies showedthis process was sensitive to both the endocytosis inhibitor,cytocholasin B, as well as to intracellar potassium-depletion,suggesting that the mechanisms of receptor-mediated endocytosis played arole in CD40 regulation (FIG. 8A). Furthermore, treatment of the D2SC/Iline with the endosomal 1-EATPase inhibitor balifomycin A₁ enhancedtotal CD40 levels based on intracellular staining assays. Overall, theseresults suggested that upon ligand engagement, CD40 was taken up byendocytosis and degraded by lysosomal proteolytic processing (FIG. 8B).Additional work indicates that the inhibition of CD40 endocytosisenhances its signaling capacity (FIG. 8C).

Expression of the truncated “type II” isoform of CD40, which lacks botha transmembrane and cytoplasmic domain, was upregulated in response toDC maturation (FIG. 9A). Previous work has shown that the type II CD40isoform abrogated surface expression, as well as total cellularexpression, of type I CD40 (Tone et al. 2001). It was hypothesized thatthis inhibitory mechanism involved homotypic interactions between theextracellular domains of the type I and II CD40 isoforms, however, nodirect evidence was presented to provide further insight into thisregulatory pathway. Therefore, it was posited that iCD40 would beresistant to type II CD40-mediated inhibition due to the absence of aligand-binding ectodomain. To investigate the potential regulation ofendogenous type I CD40 and iCD40 by this alternatively spliced geneproduct, the type II CD40 isoform was rt-PCR-amplified from purifiedbone marrow-derived murine DCs and sub-cloned into a ZeoR-expressingvector in-frame with the c-myc-derived epitope tag. This construct wasused to generate clonal “double-stable” DCs that expressed both iCD40and type II CD40 (IICD40) by selecting cell lines in G418 andzeocin-containing media. High (hi), intermediate (int), and low (10)IICD40-expressing DC lines were selected by anti-myc western blotanalysis for further study (FIG. 9B). An anti-HA blot of these sameiCD40-IICD40 DC lines demonstrated that the total cellular expression ofiCD40 was not affected by over-expression of the IICD40 transgene (FIG.9B). However, flow cytometry analysis of type I CD40 surface expressiondemonstrated a reduction in the mean fluorescence intensity of thisreceptor in IICD40-expressing DC lines (FIG. 9C). Moreover, thepredicted inverse relationship was found to exist between the expressionlevel of type II CD40 and the expression of type I CD40. These data wasconsistent with that of previous work that demonstrated IICD40-mediatedtype I CD40 downregulation in a macrophage cell line.

To expand upon these findings, the effects of CD40L and AP20187titrations on each of the IICD40 lines were analyzed based on thehypothesis that IICD40-mediated type I CD40 downregulation should bluntthe DC response to CD40L but not to AP20187 (FIG. 9D). Using MHC class IH-2K^(d) surface expression as a reporter, the results revealed thatIICD40 shifted the dose-response curve such that elevated amounts ofCD40L were required to initiate signaling through the CD40 axis while,in contrast, AP20187 induced even higher levels of H-2K^(d) than theempty vector control. Overall, these data confirmed the findings of Toneet al. and supported the notion that type I CD40-IICD40 interactionsoccurred via the homologous extracellular domains of these receptors.Furthermore, these results suggested that the iCD40 receptor switch wascapable of circumventing negative feedback regulatory mechanismsinvolving dominant negative CD40 isoforms.

Example 16 Generation of a CD11c Construct in Order to Generate aDC-Specific iCD40 Transgenic Mouse

To generate a DC-specific iCD40-expressing transgenic mouse. iCD40 wassubcloned into a vector containing the DC-specific CD11c promoter andpurified by CsCl gradient ultracentrifugation. After sequencing, thevector was shown to induce the expression of iCD40 in 293T cells. DNAmicroinjections were performed in C57BL/6 mice.

Bone marrow-derived DCs were isolated from PCR-positive offspring foranti-HA western blots. Although iCD40 protein expression was found inall mice, the levels varied from barely detectable to easily detectable.These mice are currently being bred for functional experiments, butearly results show pronounced expansion of splenic DCs iniCD40-expressing mice, with increased activation of T cells followingCID administration.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method for inducing an immune response in vivo,which comprises administering to a subject a composition comprising anucleic acid comprising a promoter operably linked to a nucleic acidsequence that encodes a chimeric protein, wherein the chimeric proteincomprises a membrane targeting region, a multimeric ligand bindingregion and a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain, whereby an immune response is induced.
 2. Themethod of claim 1, further comprising administering a nucleic acidcomprising a nucleotide sequence encoding an antigen.
 3. The method ofclaim 1, wherein the composition further comprises a nucleic acidcomprising a promoter operably linked to a nucleotide sequence encodingan antigen.
 4. The method of claim 1, wherein the composition isdelivered by injection or intravenously.
 5. The method of claim 1,wherein the composition is delivered intradermally or subcutaneously. 6.The method of claim 1, further comprising administering a ligand thatbinds to the ligand binding region
 7. The method of claim 1, wherein themembrane targeting region is selected from the group consisting of amyristoylation region, palmitoylation region, prenylation region, andtransmembrane sequences of receptors.
 8. The method of claim 1, whereinthe membrane targeting region is a myristoylation region.
 9. The methodof claim 1, wherein the multimeric ligand binding region is selectedfrom the group consisting of FKBP, cyclophilin receptor, steroidreceptor, tetracycline receptor, heavy chain antibody subunit, lightchain antibody subunit, single chain antibodies comprised of heavy andlight chain variable regions in tandem separated by a flexible linkerdomain, and mutated sequences thereof.
 10. The method of claim 1,wherein the multimeric ligand binding region is an FKBP12 region. 11.The method of claim 9, wherein the FKBP region is Fv'Fvls.
 12. Themethod of claim 1, wherein the multimeric ligand is an FK506 dimer or adimeric FK506 analog ligand.
 13. The method of claim 1, wherein theligand is AP1903.
 14. The composition of claim 1, wherein the nucleicacid is contained within a viral vector.
 15. The composition of claim14, wherein the viral vector is an adenoviral vector.
 16. Thecomposition of claim 1, wherein the nucleic acid is contained within aplasmid vector.
 17. The composition of claim 2, wherein the antigen is atumor antigen.
 18. The composition of claim 2, wherein the antigen is aprostate cancer antigen.